Neutralizing anti-influenza a virus antibodies and uses thereof

ABSTRACT

The invention relates to antibodies, and antigen binding fragments thereof, that specifically bind to an epitope in the stem region of an influenza A hemagglutinin trimer and neutralize a group 1 subtype and a group 2 subtype of influenza A virus. The invention also relates to nucleic acids that encode, immortalized B cells and cultured single plasma cells that produce, and to epitopes that bind to such antibodies and antibody fragments. In addition, the invention relates to the use of the antibodies, antibody fragments, and epitopes in screening methods as well as in the diagnosis, treatment and prevention of influenza A virus infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/233,719, filed under 35 U.S.C. §371 as the U.S. national phase ofInternational Application No. PCT/IB2011/002329, filed Jul. 18, 2011,which designated the United States, each of which is hereby incorporatedby reference in its entirety including all tables, figures, and claims.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 19, 2017, isnamed 304.0031USC2_SeqListing.txt and is 25 kilobytes in size.

BACKGROUND

The neutralizing antibody response to Influenza A virus is typicallyspecific for a given viral subtype. There are 16 influenza A subtypesdefined by their hemagglutinin (“HA”) proteins. The 16 HAs, H1-H16, canbe classified into two groups. Group 1 consists of H1, H2, H5, H6, H8,H9, H11, H12, H13, and H16 subtypes, and group 2 includes H3, H4, H7,H10, H14 and H15 subtypes. While all subtypes are present in birds,mostly H1, H2 and H3 subtypes cause disease in humans. H5, H7 and H9subtypes are causing sporadic severe infections in humans and maygenerate a new pandemic. H1 and H3 viruses continuously evolvegenerating new variants, a phenomenon called antigenic drift. As aconsequence, antibodies produced in response to past viruses are poorly-or non-protective against new drifted viruses. A consequence is that anew vaccine has to be produced every year against H1 and H3 viruses thatare predicted to emerge, a process that is very costly as well as notalways efficient. The same applies to the production of a H5 influenzavaccine. Indeed it is not clear whether the current H5 vaccines based onthe Vietnam or Indonesia influenza A virus isolates will protect againsta future pandemic H5 virus.

For these reasons it would be highly desirable to have a vaccine thatinduces broadly neutralizing antibodies capable of neutralizing allinfluenza A virus subtypes as well as their yearly variants (reviewed byGerhard et al., 2006). In addition broadly neutralizing heterosubtypicantibodies could be administered as medicaments for prevention ortherapy of influenza A infection. For the manufacture of suchmedicaments it is important to select antibodies that are produced athigh titers to reduce costs of production.

Antibodies that recognize influenza A virus have been characterized.Antibodies to M2, an invariant small protein expressed on infected cellsbut not on infectious viruses, have shown some protective effect invivo, possibly by targeting infected cells for destruction by NK cellsor cytotoxic T cells. It is also possible to target the HA protein withneutralizing antibodies. HA is synthesized as a homo-trimeric precursorpolypeptide HA0. Each monomer can be independently cleavedpost-translationally to form two polypeptides, HA1 and HA2, linked by asingle disulphide bond. The larger N-terminal fragment (HA1, 320-330amino acids) forms a membrane-distal globular domain that contains thereceptor-binding site and most determinants recognized byvirus-neutralizing antibodies. The HA1 polypepetide of HA is responsiblefor the attachment of virus to the cell surface. The smaller C-terminalportion (HA2, ≈180 amino acids) forms a stem-like structure that anchorsthe globular domain to the cellular or viral membrane. The HA2polypeptide mediates the fusion of viral and cell membranes inendosomes, allowing the release of the ribonucleoprotein complex intothe cytoplasm.

The degree of sequence homology between subtypes is smaller in the HA1polypeptides (34%-59% homology between subtypes) than in the HA2polypeptide (51%-80% homology). The most conserved region is thesequence around the cleavage site, particularly the HA2 N-terminal 11amino acids, termed fusion peptide, which are conserved among allinfluenza A virus subtypes. Part of this region is exposed as a surfaceloop in the HA precursor molecule (HA0), but becomes inaccessible whenHA0 is cleaved into HA1/HA2. In summary there are conserved regionsamong different HA subtypes especially in the HA1-HA2 joining region andin the HA2 region. However these regions may be poorly accessible toneutralizing antibodies.

There has only been limited success in identifying antibodies thatneutralize more than one subtype of influenza A virus. Further, thebreath of neutralization of antibodies identified thus far is narrow andtheir potency is low. Okuno et al, immunized mice with influenza virusA/Okuda/57 (H2N2) and isolated a monoclonal antibody (C179) that bindsto a conserved conformational epitope in HA2 and neutralizes the Group 1H2, H1 and H5 subtype influenza A viruses in vitro and in vivo in animalmodels (Okuno et al.,1993; Smirnov et al., 1999; Smirnov et al., 2000).

Gioia et al., described the presence of H5N1 virus neutralizingantibodies in the serum of some individuals that received a conventionalseasonal influenza vaccine (Gioia et al., 2008). The authors suggestthat the neutralizing activity might be due to antibodies toneuraminidase (N1). However, monoclonal antibodies were not isolated andtarget epitopes were not characterized. Also, it is not clear whetherthe serum antibodies neutralize other subtypes of influenza A virus.

Heterosubtypic human antibodies that bind to an epitope in the stem-likeregion of HA, and capable of neutralizing some influenza A virussubtypes within either Group 1 or Group 2, have been isolated frommemory B cells and plasma cells of immune donors. However, InfluenzaA—specific neutralizing antibodies targeting epitopes in the HA trimerconserved on all 16 subtypes and capable of neutralizing viruses of bothGroup 1 and Group 2 subtypes have not been found so far, and theirisolation remains a major goal for therapeutic approaches and vaccinedesign.

Despite decades of research, there are no marketed antibodies thatbroadly neutralize or inhibit influenza A virus infection or attenuatedisease caused by influenza A virus. Therefore, there is a need toidentify new antibodies that neutralize multiple subtypes of influenza Avirus and can be used as medicaments for prevention or therapy ofinfluenza A infection. There is a further need to identify antibodiesthat are produced at high titers to reduce costs of production.

SUMMARY

The invention is based, in part, on the isolation from individualsvaccinated with the seasonal influenza vaccine of naturally occurringhuman monoclonal antibodies that bind to HA and neutralize infection ofmore than one subtype of influenza A virus, as well as novel epitopes towhich the antibodies of the invention bind. Accordingly, in one aspectof the invention, the invention comprises an antibody and antigenbinding fragments thereof that neutralize infection of more than onesubtype of influenza A virus, selected from group 1 and group 2subtypes.

In one embodiment of the invention, the invention comprises an isolatedantibody, or an antigen binding fragment thereof, that neutralizesinfection of a group 1 subtype and a group 2 subtype of influenza Avirus. In another embodiment of the invention, it comprises an isolatedantibody, or an antigen-binding fragment thereof, that neutralizesinfection of a group 1 subtype and a group 2 subtype of influenza Avirus and specifically binds to an epitope in the stem region of aninfluenza A hemagglutinin (HA) trimer, wherein the heavy and light chainof the antibody, or antigen binding fragment thereof, contact aminoacids in a first, proximal monomer and a second, distal, right monomerof the HA trimer.

In yet another embodiment of the invention, the invention comprises anisolated antibody, or an antigen-binding fragment thereof, thatneutralizes infection of a group 1 subtype and a group 2 subtype ofinfluenza A virus and specifically binds to an epitope in the stemregion of an influenza A HA trimer, wherein the heavy and light chain ofthe antibody, or antigen binding fragment thereof, contact amino acidsin a first, proximal monomer and a second, distal, right monomer of theHA trimer, and wherein the antibody, or antigen-binding fragmentthereof, is produced in transfected cells at titers of at least 3 foldhigher than the titer at which FI6 variant 2 is produced.

In still another embodiment of the invention, the invention comprises anisolated antibody, or an antigen-binding fragment thereof, thatneutralizes infection of a group 1 subtype and a group 2 subtype ofinfluenza A virus and specifically binds to an epitope in the stemregion of an influenza A HA trimer, wherein the heavy and light chain ofthe antibody, or antigen binding fragment thereof, contact amino acidsin a first, proximal monomer and a second, distal, right monomer of theHA trimer, and wherein the antibody, or antigen-binding fragmentthereof, comprises: (i) the heavy chain CDR1, CDR2 and CDR3 sequences asset forth in SEQ ID NOs: 1, 41 and 43, respectively, or as set forth inSEQ ID NOs: 1, 41 and 42, respectively; and (ii) the light chain CDR1,CDR2, and CDR3 sequences as set forth in SEQ ID NOs: 4, 5 and 6,respectively, or as set forth in SEQ ID NOs: 44, 5 and 6, respectively.

In another embodiment of the invention, the invention comprises anisolated antibody, or an antigen binding fragment thereof, comprising atleast one complementarity determining region (CDR) sequence having atleast 95% sequence identity to any one of SEQ ID NOs: 1-6, 17-22, or41-44, wherein the antibody neutralizes influenza A virus.

In another embodiment of the invention, it comprises an isolatedantibody, or an antigen-binding fragment thereof, that neutralizesinfection of a group 1 subtype and a group 2 subtype of influenza Avirus and comprises: (i) the heavy chain CDR1, CDR2 and CDR3 sequencesas set forth in SEQ ID NOs: 1, 41 and 43, respectively, or as set forthin SEQ ID NOs: 1, 41 and 42, respectively; and (ii) the light chainCDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NOs: 4, 5 and 6,respectively, or as set forth in SEQ ID NOs: 44, 5 and 6, respectively.

In yet another embodiment of the invention, the invention comprises anisolated antibody, or an antigen binding fragment thereof, comprising aheavy chain CDR1 with the amino acid sequence of SEQ ID NO: 1 or SEQ IDNO: 17; a heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 18, or SEQ ID NO: 41; and a heavy chain CDR3 with the aminoacid sequence of SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 42 or SEQ IDNO: 43, wherein the antibody neutralizes influenza A virus. In yetanother embodiment of the invention, it comprises an isolated antibody,or an antigen binding fragment thereof, comprising a light chain CDR1with the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 20 or SEQ IDNO: 44; a light chain CDR2 with the amino acid sequence of SEQ ID NO: 5or SEQ ID NO: 21; and a light chain CDR3 with the amino acid sequence ofSEQ ID NO: 6 or SEQ ID NO: 22, wherein the antibody neutralizesinfluenza A virus.

In still another embodiment of the invention, the invention comprises anisolated antibody, or an antigen binding fragment thereof, wherein theantibody comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 13 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 14; or a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 33 anda light chain variable region comprising the amino acid sequence of SEQID NO: 14; or a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 29 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 30; or a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 35 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:30; or a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 59 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 57; or a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 59 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 61; ora heavy chain variable region comprising the amino acid sequence of SEQID NO: 55 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 57; or a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 55 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 61 and whereinthe antibody neutralizes a group 1 subtype and a group 2 subtype ofinfluenza A virus. The invention further comprises an antibody, or anantigen binding fragment thereof, wherein the antibody is FI6 variant 1,FI6 variant 2, FI6 variant 3, FI6 variant 4, or FI6 variant 5.

In yet another embodiment of the invention, the invention comprises anantibody, or antigen binding fragment thereof, that neutralizesinfection of a group 1 subtype and a group 2 subtype of influenza Avirus, wherein the antibody or fragment thereof is expressed by animmortalized B cell clone.

In another aspect, the invention comprises a nucleic acid moleculecomprising a polynucleotide encoding an antibody or antibody fragment ofthe invention. In yet another aspect, the invention comprises a vectorcomprising a nucleic acid molecule of the invention or a cell expressingan antibody of the invention or an antigen binding fragment thereof. Inyet another embodiment, the invention comprises a cell comprising avector of the invention. In still another aspect, the inventioncomprises an isolated or purified immunogenic polypeptide comprising anepitope that binds to an antibody or antigen binding fragment of theinvention.

The invention further comprises a pharmaceutical composition comprisingan antibody of the invention or an antigen binding fragment thereof, anucleic acid molecule of the invention, a vector comprising a nucleicacid molecule of the invention, a cell expressing an antibody or anantibody fragment of the invention, a cell comprising a vector of theinvention, or an immunogenic polypeptide of the invention, and apharmaceutically acceptable diluent or carrier. The invention alsocomprises a pharmaceutical composition comprising a first antibody or anantigen binding fragment thereof, and a second antibody, or an antigenbinding fragment thereof, wherein the first antibody is an antibody ofthe invention, and the second antibody is any antibody, or antigenbinding fragment thereof, that neutralizes influenza A or influenza Bvirus infection.

Use of an antibody of the invention, or an antigen binding fragmentthereof, a nucleic acid of the invention, a vector comprising a nucleicacid of the invention, a cell expressing a vector of the invention, anisolated or purified immunogenic polypeptide comprising an epitope thatbinds to an antibody or antibody fragment of the invention, or apharmaceutical composition of the invention (i) in the manufacture of amedicament for the treatment of influenza A virus infection, (ii) in avaccine, or (iii) in diagnosis of influenza A virus infection is alsocontemplated to be within the scope of the invention. Further, use of anantibody of the invention, or an antigen binding fragment thereof, formonitoring the quality of anti-influenza A virus vaccines by checkingthat the antigen of said vaccine contains the specific epitope in thecorrect conformation is also contemplated to be within the scope of theinvention.

In another aspect, the invention provides a method of preventing,treating or reducing influenza A virus infection or lowering the risk ofinfluenza A virus infection comprising administering to a subject inneed thereof, a therapeutically effective amount of an antibody or anantigen binding antibody fragment of the invention.

In a further aspect, the invention comprises an epitope whichspecifically binds to an antibody of the invention, or an antigenbinding fragment thereof, for use (i) in therapy, (ii) in themanufacture of a medicament for treating influenza A virus infection,(iii) as a vaccine, or (iv) in screening for ligands able to neutraliseinfluenza A virus infection.

DESCRIPTION OF FIGURES

FIG. 1 shows the titers of antibody production of 293F cells transientlytransfected with vectors expressing genes encoding FI6 variant 2, 3. 4or 5.

FIG. 2A is a surface representation of the F subdomain of H1 HA incomplex with FI6 variant 3 (referred to in the figure as FI6). FIG. 2Bis a surface representation of the F subdomain of and H3 HA in complexwith FI6 variant 3 (referred to in the figure as FI6). The selectedside-chains in HA1 and HA2 that contribute to the conserved hydrophobicgroove are indicated by the arrows, the approximate boundaries of whichare indicated by the black line. Thr (40) and Thr (318) are in HA1 andIle (45), Trp (21), Thr (41), Leu (38) and the 18-21 turn are in HA2.

FIGS. 3A-3E show the binding of FI6 variant 3 (referred to in the figureas FI6) to the F subdomain of the HA trimer. FIG. 3A shows the trimer ofH3 HA binding three FI6 variant 3 antibodies in Ribbons representation.One of the HA monomers is colored black for HA1 and dark grey for HA2,while the other two HA monomers are in light grey. FIG. 3B shows a zoomview of the interaction of the LCDR1 loop with the fusion peptide of theneighboring HA monomer in H1/FI6 variant 3 complexes. FIG. 3C shows azoom view of the interaction of the LCDR1 loop with the fusion peptideof the neighboring HA monomer in H3/FI6 variant 3 complexes. FIG. 3Dshows the structures of monomers from H5 HA in complex with CR6261 (pdbID 2GBM) binding to a similar region compared to FI6 variant 3 but withtheir VH domain sitting 5-10 Å lower on the HA. FIG. 3E show thestructures of monomers from H5 HA in complex with F10 (pdb 3FKU) bindingto a similar region compared to FI6 variant 3 but with their VH domainsitting 5-10 Å lower on the HA.

FIG. 4 shows the interactions of FI6 variant 3 (referred to in thefigure as FI6-v3) with the F sub-domain of H1 and H3 HA. It depicts asurface representation of the F subdomain of H1 HA and H3 HA with theHCDR3 and LCDR1 loops of FI6 variant 3 with selected side chains. Theproximal HA monomer is depicted in light grey; the distal right monomeris depicted in light grey; and the glycan bound to N38 of H3 HA1 isdepicted as dark grey spheres.

FIGS. 5A-5D show group-specific differences at the cross-reactiveantibody binding sites. FIG. 5A shows the positioning of thecarbohydrate side chain at Asn-38 in the H3 HA apo-structure. FIG. 5Bshows the positioning of the carbohydrate side chain at Asn-38 in theFI6 variant 3 bound structure (B). FIGS. 5C and 5D show the orientationof HA1 Trp-21 in different antibody complexes. FIG. 5C shows Phe-100D ofthe HCDR3 loop of FI6 variant 3 (referred to in the figure as FI6)interacting with the F sub-domain of the H1 or H3 HA. FIG. 5D shows theHCDR2 loop of the F10 and CR6261 antibodies presenting Phe-55 and Phe-54respectively, towards Trp-21 of H5 HA.

FIGS. 6A-6D show the contact surface of FI6 variant 3 on HA. The fourpanels show the footprints (contoured with a black line) on HA of FI6variant 3, CR6261 and F10 antibodies; the three HA monomers are depictedas white, light or dark grey. FIG. 6A:

Contact footprint for FI6 variant 3/H1 uncleaved; FIG. 6B: FI6 variant3/H3 cleaved; FIG. 6C: CR6261/H5; and FIG. 6D: F10/H5. Unlike CR6261 andF10, FI6 variant 3 makes contact with two HA monomers. For the FI6variant 3 complexes, glycosylation sites at the antibody/HA interfaceare labeled.

FIG. 7 shows the residues (in bold, Kabat numbering) contacting HA inFI6 variant 3 VH and VK chains.

FIGS. 8A-G show that FI6 variant 3 confers protection in mouse models ofInfluenza A virus infection. FIG. 8A shows survival curves and FIG. 8Bshows the body weight loss of BALB/c mice (five per experimentalcondition) that received different doses of FI6 variant 3 i.v. threehours before intranasal infection with 10 MLD50 (50% lethal dose inmice) H1N1 A/PR/8/34 virus. FIG. 8C shows the body weight loss of mice(ten per experimental condition) that received different doses of FI6variant 3 i.v. three hours before infection with 3×10⁵ pfu of H3N2HK-x31 virus. Shown is one representative experiment out of the threethat were performed. FIG. 8D shows survival curve and FIG. 8E shows bodyweight loss of mice (five per experimental condition) that received 15mg/kg of FI6 variant 3 i.v. on day 0 (3 hours before infection) or onday 1, 2 and 3 after infection with 10 MLD50 of A/PR/8/34 virus. Onerepresentative experiment out of the two performed is shown. Shown aremean values ±SD. FIG. 8F shows survival curves of mice (ten perexperimental condition) that received 10 mg/kg of FI6 variant 2(FI6-v2), FI6-v2 KA (that lack complement binding), FI6-v2 LALA (thatlack complement and FcR binding) or control antibody one day beforeinfection with A/PR/8/34 virus. FIG. 8G shows survival curves of mice(ten per experimental condition) that received 3 mg/kg of FI6 variant 2(FI6-v2), FI6-v2 KA (that lack complement binding), FI6-v2 LALA (thatlack complement and FcR binding) or control antibody one day beforeinfection with A/PR/8/34 virus.

FIG. 9 shows pulmonary virus titers of mice treated with FI6 variant 3after H1N1 PR/8/34 lethal challenge. BALB/c mice (four mice perexperimental condition) received i.v. injection of 15 mg/kg FI6 variant3 or a control antibody (HJ16, HIV-1 specific) three hours before (day0) or 1, 2 or 3 days after i.n. infection with 10 MLD50 H1N1PR/8/34.Viral titers were determined 4 days post infection in lungs. Virus wasundetectable in the brains. Data are displayed in box-and-whiskers formin which the box extends from the 25th to the 75th percentile, with ahorizontal line at the median. Whiskers above and below the box indicatethe extreme values. Results of Students' t-test statistic analysis arenoted as * for p<0.05, and as *** for p<0.001.

FIG. 10 shows that FI6 variant 3 binding to HA stem region interfereswith protease-mediated HA0 cleavage. Recombinant HA from the H1 NC/99isolate was incubated with FI6 variant 3, FE17 (a human antibody thatrecognizes the Ca2 site on the HA globular head) or a control antibody.The HA-antibody mixture was then exposed to TPCK-treated trypsin for 5,10 or 20 minutes at 37° C. The samples were then run on a polyacrilamidegel and Western blots were developed using a biotinylated human mAb(FO32) that recognizes HA2 and HA0 of all influenza A strains underdenaturing conditions. Shown is the HA0 band. One representativeexperiment out of three is shown.

DETAILED DESCRIPTION

The invention is based, in part, on the discovery and isolation, fromindividuals that were vaccinated with the seasonal influenza A vaccine,of naturally occurring human antibodies that broadly neutralizeinfluenza A virus of different subtypes as well as novel epitopes towhich the antibodies of the invention bind. Such antibodies aredesirable, as only one or few antibodies are required in order toneutralize different subtypes of influenza A virus. Further, the broadlyneutralizing heterosubtypic antibodies are produced at high titers toreduce costs of production of medicaments comprising the antibodies. Inaddition, the epitopes recognized by such antibodies may be part of avaccine capable of inducing broad protection against both seasonal andcandidate pandemic isolates of different subtypes.

Accordingly, in one aspect, the invention provides an isolated antibody,and antigen binding fragments thereof, that neutralize at least twoinfluenza A viruses in group 1 and group 2 subtypes. In one embodiment,the invention provides an isolated antibody, or an antigen bindingfragment thereof, that neutralizes infection of a group 1 subtype and agroup 2 subtype of influenza A virus.

In another embodiment, it provides an isolated antibody, or anantigen-binding fragment thereof, that neutralizes infection of a group1 subtype and a group 2 subtype of influenza A virus and specificallybinds to an epitope in the stem region of an influenza A HA trimer,wherein the heavy and light chain of the antibody, or antigen bindingfragment thereof, contact amino acids in a first, proximal monomer and asecond, distal, right monomer of the HA trimer.

As discussed earlier, the HA protein is synthesized as a trimericprecursor polypeptide HA0 comprising three identical monomers(homo-trimer). Each monomer may, or may not, be cleaved independent ofthe other two monomers. Upon post-translational cleavage, each monomerforms two polypeptides, HA1 and HA2, which are otherwise linked by asingle disulphide bond. The heavy and light chains of the antibodies ofthe invention contact two of the three monomers of the HA trimer. Themonomers contacted by the antibodies of the invention may be cleaved, oruncleaved. For clarity purposes and for the purposes of understandingthe schematic representations in the figures, we refer to the twomonomers contacted by the antibodies of the invention as the proximalmonomer and the distal, right monomer.

As used herein, the terms “antigen binding fragment,” “fragment,” and“antibody fragment” are used interchangeably to refer to any fragment ofan antibody of the invention that retains the antigen-binding activityof the antibody. Examples of antibody fragments include, but are notlimited to, a single chain antibody, Fab, Fab′, F(ab′)2, Fv or scFv.Further, the term “antibody” as used herein includes both antibodies andantigen binding fragments thereof.

As used herein, a “neutralizing antibody” is one that can neutralize,i.e., prevent, inhibit, reduce, impede or interfere with, the ability ofa pathogen to initiate and/or perpetuate an infection in a host. Theterms “neutralizing antibody” and “an antibody that neutralizes” or“antibodies that neutralize” are used interchangeably herein. Theseantibodies can be used, alone or in combination, as prophylactic ortherapeutic agents upon appropriate formulation, in association withactive vaccination, as a diagnostic tool, or as a production tool asdescribed herein.

X-ray crystallography studies of Group-1 specific heterosubtypicantibodies co-crystallized with H1 and H5 HA known in the art show thatthe antibodies interact with only one monomer of the HA trimer. Further,the studies show that the antibodies contact the HA with only the CDRresidues of the heavy chain but not of the light chain. In contrast, theantibodies or antigen binding fragments of the invention contact, notone, but two of the three HA monomers. Additionally, the antibodies ofthe invention contact the HA with CDR residues from both the heavy chainand the light chain. In addition, the nature of the interactions made byantibodies or antigen binding fragments of the invention with the HA aremarkedly different to those made by the other antibodies, CR6261 andF10. The most striking difference is that the interaction of theantibodies of the invention with the hydrophobic groove on HA ismediated solely by CDR3 of the heavy chain (HCDR3), whereas for CR6261and F10 all three HCDRs are involved in the binding.

In one embodiment, the heavy chains of the antibodies or antigen bindingfragments of the invention contact amino acid residues in the proximalmonomer, and the light chains of the antibodies or antigen bindingfragments of the invention contact amino acid residues in both theproximal monomer and in the distal right monomer of the HA trimer. Themonomers contacted by the antibodies of the invention, i.e., theproximal monomer and the distal right monomer, may be uncleaved or theymay be cleaved to form the HA1 and HA2 polypeptides. In one embodiment,the proximal monomer and the distal right monomer are cleaved. Inanother embodiment, the proximal monomer and the distal right monomerare uncleaved.

The antibodies and antigen-binding fragments of the inventionspecifically bind to an epitope that is conserved amongst the 16different HAs of the 16 subtypes of influenza A virus. In oneembodiment, the antibodies or antigen binding fragments of the inventionbind to an epitope that comprises the amino acid residue at position 329of HA1 and the amino acid residues at positions 1, 2, 3, and 4 of HA2,wherein the HA1 and HA2 are present in an uncleaved monomer of the HAtrimer.

In another embodiment, the heavy chains of the antibodies or antigenbinding fragments of the invention contact the amino acid residue atposition 318 in HA1 and amino acid residues at positions 18, 19, 20, 21,38, 41, 42, 45, 49, 53, and 57 in HA2 of either the proximal or thedistal right monomer. The monomers may be uncleaved or cleaved.

In yet another embodiment, the light chains of the antibodies or antigenbinding fragments of the invention contact amino acid residues atpositions 38, 39, and 43 in HA2 of the uncleaved, proximal monomer, andamino acid residues at positions 327, 328, and 329 in HA1 and 1, 2, 3,and 4 in HA2 of the uncleaved, distal right monomer.

In still another embodiment, the antibodies and antigen-bindingfragments of the invention specifically bind to an epitope thatcomprises the amino acid residue at position 318 of the HA1 and theamino acid residues at positions 18, 19, 20, 21, 38, 39, 41, 42, 43, 45,48, 49, 53, 56, and 57 of the HA2 of the uncleaved, proximal monomer. Inaddition, the antibodies specifically bind to an epitope that comprisesthe amino acid residues at positions 327, 328, 329 of the HA1 and theamino acid residues at positions 1, 2, 3, and 4 of the HA2 polypeptideof the uncleaved, distal right monomer.

In another embodiment, the light chains of the antibodies or antigenbinding fragments of the invention contact amino acid residues atpositions 38, 39, 42, and 46 in HA2 of the proximal monomer and aminoacid residues at positions 321 and 323 in HA1 and 7 and 11 in HA2 of thedistal right monomer. In this embodiment both the proximal and distalright monomers are cleaved.

In yet another embodiment, the antibodies and antigen-binding fragmentsof the invention specifically bind to an epitope that comprises theamino acid residue at position 318 of the HA1 and the amino acidresidues at positions 18, 19, 20, 21, 38, 39, 41, 42, 45, 46, 49, 52,53, and 57 of HA2 of the cleaved proximal monomer, as well as the aminoacid residues at positions 321 and 323 of HA1 and amino acid residues atpositions 7 and 11 of HA2 of the cleaved distal right monomer.

As shown herein, the antibodies or antigen binding fragments of theinvention are capable of binding specifically to the HAs of all 16subtypes of influenza A virus. In one embodiment, the antibodies of theinvention specifically bind to an influenza A HA of subtypes H1, H2, H3,H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16.

In another embodiment of the invention, the invention providesantibodies that have high titers of production. As an example, once twovery similar antibodies of the invention, FI6 variant 1 and FI6 variant2, were isolated, several variants of the antibodies (in particular,variants of FI6 variant 2) were synthesized to improve production intransfected cells. In one embodiment, antibodies or antigen bindingfragments of the invention are produced in transfected cells at titersof at least 1.5 fold higher than the titer at which FI6 variant 2 isproduced. In another embodiment, the antibodies of the invention areproduced at titers of at least 1.8, 2, 2.2, 2.5, 2.7, 3, 3.2, 3.4, 3.6,3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.3, 5.6 or 6 fold higher than the titerat which FI6 variant 2 is produced. In some embodiments, the antibodiesor antigen binding fragments of the invention are produced intransfected cells at titers of at least 3, at least 4, or at least 4.5fold higher than the titer at which FI6 variant 2 is produced.

Thus, in one embodiment, the invention provides an isolated antibody, oran antigen-binding fragment thereof, that neutralizes infection of agroup 1 subtype and a group 2 subtype of influenza A virus andspecifically binds to an epitope in the stem region of an influenza A HAtrimer, wherein the heavy and light chain of the antibody, or antigenbinding fragment thereof, contact amino acids in a first, proximalmonomer and a second, distal, right monomer of the HA trimer, andwherein the antibody, or antigen-binding fragment thereof, is producedin transfected cells at titers higher, for example, at least 3 foldhigher, than the titer at which FI6 variant 2 is produced.

As described herein, the transfected cells may be any cells now knownto, or later discovered by one of skill in the art for expressing thenucleic acid sequences encoding the antibodies of the invention.Examples of such cells include, but are not limited to, mammalian hostcells such as CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells.Further, the cells may be transfected either transiently or stably. Thetype of transfection as well as the cell type suitable for use intransfection is within the scope of one of skill in the art.

In another embodiment, the antibody, or antigen binding fragments of theinvention, specifically binds to a polypeptide comprising the amino acidsequence of any one of SEQ ID NOs: 37, 38, 39 or 40.

Human monoclonal antibodies, the immortalized B cell clones or thetransfected host cells that secrete antibodies of the invention, andnucleic acid encoding the antibodies of the invention are also includedwithin the scope of the invention. As used herein, the term “broadspecificity” is used to refer to an antibody or an antigen bindingfragment of the invention that can bind and/or neutralize one or moregroup 1 subtype and one or more group 2 subtype of influenza A virus.

The antibody, or antigen binding fragments, of the invention neutralizesone or more influenza A virus from group 1 (H1, H2, H5, H6, H8, H9, H11,H12, H13, and H16 and their variants) and one or more influenza A virusfrom group 2 (H3, H4, H7, H10, H14 and H15 and their variants) subtypes.In one embodiment, exemplary group 1 subtypes include H1, H2, H5, H6,and H9 and exemplary group 2 subtypes include H3 and H7.

The antibody and antibody fragment of the invention is capable ofneutralizing various combinations of influenza A virus subtypes. In oneembodiment, the antibody can neutralize influenza A virus H1 and H3subtypes, or H2 and H3 subtypes, or H3 and H5 subtypes, or H3 and H9subtypes, or H1 and H7 subtypes, or H2 and H7 subtypes, or H5 and H7subtypes, or H7 and H9 subtypes.

In another embodiment, the antibody and antibody fragment of theinvention can neutralize influenza A virus H1, H2 and H3 subtypes, orH1, H3 and H5 subtypes, or H1, H3 and H9 subtypes, or H2, H3 and H5subtypes, or H2, H3 and H9 subtypes, or H3, H5 and H9 subtypes, or H1,H2 and H7 subtypes, or H1, H5 and H7 subtypes, or H1, H7 and H9subtypes, or H2, H5 and H7 subtypes, or H2, H7 and H9 subtypes, or H5,H7 and H9 subtypes, or H1, H3 and H7 subtypes, or H2, H3 and H7subtypes, or H3, H5 and H7 subtypes, or H3, H7 and H9 subtypes.

In yet another embodiment, the antibody can neutralize influenza A virusH1, H2, H3 and H7 subtypes, or H1, H3, H5 and H7 subtypes, or H1, H3, H7and H9 subtypes, or H2, H3, H5 and H7 subtypes, or H2, H3, H7 and H9subtypes, or H3, H5, H7 and H9 subtypes or H1, H2, H3 and H5 subtypes,or H1, H2, H3 and H9 subtypes, or H1, H3, H5 and H9 subtypes, or H2, H3,H5 and H9 subtypes, or H1, H2, H5 and H7 subtypes, or H1, H2, H7 and H9subtypes, or H1, H5, H7 and H9 subtypes, or H2, H5, H7 and H9 subtypes.

In still another embodiment, the antibody of the invention canneutralize influenza A virus H1, H2, H3, H5 and H7 subtypes, or H1, H2,H3, H7 and H9 subtypes, or H1, H3, H5, H7 and H9 subtypes, or H2, H3,H5, H7 and H9 subtypes, or H1, H2, H3, H5 and H9 subtypes, or H1, H2,H5, H7 and H9 subtypes, or H1, H2, H3, H5, H7 and H9 subtypes. In yetanother embodiment, the antibody and antigen binding fragments of theinvention neutralize one or more of the above combinations in additionto neutralizing influenza A virus H6 subtype.

The antibody and antigen binding fragment of the invention have highneutralizing potency. The concentration of the antibody of the inventionrequired for 50% neutralization of influenza A virus, can, for example,be about 50 μg/ml or less. In one embodiment, the concentration of theantibody of the invention required for 50% neutralization of influenza Avirus is about 50, 45, 40, 35, 30, 25, 20, 17.5, 15, 12.5, 11, 10, 9, 8,7, 6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 or about 1 μg/ml or less. Inanother embodiment, the concentration of the antibody of the inventionrequired for 50% neutralization of influenza A virus is about 0.9, 0.8,0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15,0.1, 0.075, 0.05, 0.04, 0.03, 0.02, 0.01, 0.008, 0.006, 0.004, 0.003,0.002 or about 0.001 μg/ml or less. This means that only lowconcentrations of antibody are required for 50% neutralization ofinfluenza A virus. Specificity and potency can be measured using astandard neutralization assay as known to one of skill in the art.

The invention provides an antibody having particularly broad specificityto HA and that neutralizes one or more influenza A virus subtypes fromgroup 1 and one or more influenza A virus subtypes from group 2. Theantibody of the invention binds to an epitope in a region of HA that isconserved among two or more influenza A virus subtypes selected fromgroup 1 and group 2.

In one embodiment, the invention provides an antibody, e.g., an isolatedantibody or a purified antibody, that specifically binds to a conservedepitope in the stem region of HA of group 1 and group 2 influenza Avirus subtypes and interferes with viral replication or spreading. Theinvention also provides an antibody, e.g., an isolated antibody or apurified antibody, that specifically binds to a conserved epitope in thestem region of HA of group 1 and group 2 subtypes and inhibits virusentry into a cell. Without being bound to any theory, in an exemplaryembodiment the antibody or antigen binding fragments of the inventionbind to a conserved epitope in the stem region of influenza A virus andinhibit virus entry into a cell by interfering with the fusion step. Inone embodiment, the antibody or antigen binding fragments of theinvention limit virus spreading by recruiting complement andFcR-expressing killer cells and mediating antibody-dependent cellcytotoxicity (ADCC). An epitope or antigenic determinant of a proteincorresponds to those parts of the molecule that are specificallyrecognized by the binding site (or paratope) of an antibody. Epitopesare thus relational entities that require complementary paratopes fortheir operational recognition. An epitope that is conserved amongdifferent variants of a protein means that the same paratope canspecifically recognize these different variants by contacting the sameparts of the molecules.

The antibodies of the invention may be monoclonal, for example, humanmonoclonal antibodies, or recombinant antibodies. The invention alsoprovides fragments of the antibodies of the invention, particularlyfragments that retain the antigen-binding activity of the antibodies.Although the specification, including the claims, may, in some places,refer explicitly to antigen binding fragment(s), antibody fragment(s),variant(s) and/or derivative(s) of antibodies, it is understood that theterm “antibody” or “antibody of the invention” includes all categoriesof antibodies, namely, antigen binding fragment(s), antibodyfragment(s), variant(s) and derivative(s) of antibodies.

In one embodiment, the antibodies and antibody fragments of theinvention neutralize a combination of two or more influenza A virussubtypes of group 1 and group 2. Exemplary influenza A virus subtypesinclude, but are not limited to, H5N1 (A/Vietnam/1203/04), H1N1 (A/NewCaledonia/20/99), H1N1 (A/Salomon Island/3/2006), H3N2 (A/Wyoming/3/03)and H9N2 (A/chicken/Hong Kong/G9/97). In another embodiment, theantibodies neutralize and/or are specific for a combination of 3, 4, 5,6, 7 or more group 1 and group 2 influenza A virus subtypes.

In an exemplary embodiment, the invention comprises an antibody, or anantibody fragment thereof, that is specific for influenza A virussubtypes H1 and H3 (e.g. H1N1 and H3N2); or H1, H3, H5, and H9 (e.g.H1N1, H3N2, H5N1 and H9N2). In yet another embodiment, the antibody orantibody fragments thereof is specific for H1, H3, H5, H7 and H9(e.g.H1N1, H3N2, H5N1, H7N1, H7N7, H9N2). Other exemplary combinationsof subtypes of influenza A virus are provided earlier in theapplication.

The SEQ ID numbers for the amino acid sequence for the heavy chainvariable region (VH) and the light chain variable region (VL) ofexemplary antibodies of the invention as well as the SEQ ID numbers forthe nucleic acid sequences encoding them are listed in Table 1.

TABLE 1 SEQ ID Numbers for V_(H) and V_(L) Polypeptides andPolynucleotides for Exemplary Influenza A Virus Neutralizing AntibodiesSEQ ID NOs. for V_(H) and V_(L) Polypeptides and Polynucleotides V_(H)V_(L) V_(H) V_(L) Poly- Poly- Polynu- Polynu- peptide peptide cleotidecleotide FI6 variant 1 13 14 15 16 FI6 variant 2 33 14 34 16 FI6 variant3 55 57 56 58 FI6 variant 4 59 57 60 58 FI6 variant 5 59 61 60 62 FI28variant 1 29 30 31 32 FI28 variant 2 35 30 36 32

In one embodiment, an antibody or antibody fragment of the inventioncomprises a heavy chain variable region having an amino acid sequencethat is about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to the sequence recited in any one of SEQ ID NOs: 13, 33, 55,59, 29 or 35. In another embodiment, an antibody or antibody fragment ofthe invention comprises a light chain variable region having an aminoacid sequence that is about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100% identical to the sequence recited in SEQ ID NOs: 14, 57, 61 or30.

In yet another embodiment, the heavy chain variable region of anantibody of the invention may be encoded by a nucleic acid that has asequence that is about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% identical to the sequence recited in any one of SEQ ID NOs: 15, 34,56, 60, 31 or 36. In yet another embodiment, the light chain variableregion of an antibody of the invention may be encoded by a nucleic acidthat has a sequence that is about 70%, 75%, 80%, 85%, 90%, 95%, 97%,98%, 99% or 100% identical to the sequence recited in SEQ ID NOs: 16,58, 62, or 32.

The CDRs of the antibody heavy chains are referred to as CDRH1 (orHCDR1), CDRH2 (or HCDR2) and CDRH3 (or HCDR3), respectively. Similarly,the CDRs of the antibody light chains are referred to as CDRL1 (orLCDR1), CDRL2 (or LCDR1) and CDRL3 (or LCDR1), respectively. Thepositions of the CDR amino acids are defined according to the IMGTnumbering system as: CDR1—IMGT positions 27 to 38, CDR2—IMGT positions56 to 65 and CDR3—IMGT positions 105 to 117.

Table 2 provides the SEQ ID numbers for the amino acid sequences of thesix CDRs of the heavy and light chains, respectively, of the exemplaryantibodies of the invention.

TABLE 2 SEQ ID Numbers for CDR Polypeptides of Exemplary Influenza AVirus Neutralizing Antibodies SEQ ID NOs. for CDR Polypeptides CDRH1CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 FI6 variant 1 1 2 3 4 5 6 FI6 variant 2 12 3 4 5 6 FI6 variant 3 1 41 42 4 5 6 FI6 variant 4 1 41 43 4 5 6 FI6variant 5 1 41 43 44 5 6 FI28 variant 1 17 18 19 20 21 22 FI28 variant 217 18 19 20 21 22

In one embodiment, an antibody or antibody fragment of the inventioncomprises at least one CDR with a sequence that has at least 95%sequence identity to any one of SEQ ID NOs: 1-6, 41-44 or 17-22,

In another embodiment, the invention provides an antibody comprising aheavy chain comprising one or more (i.e. one, two or all three) heavychain CDRs from FI6 variant 1, FI6 variant 2, FI6 variant 3, FI6 variant4, FI6 variant 5, FI28 variant 1 or FI28 variant 2. In an exemplaryembodiment, the antibody or antigen binding fragment of the inventioncomprises a heavy chain CDR1 with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO: 17; a heavy chain CDR2 with the amino acid sequence ofSEQ ID NO: 2, SEQ ID NO: 41 or SEQ ID NO: 18; and a heavy chain CDR3with the amino acid sequence of SEQ ID NO: 3, SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO: 19. In certain embodiments, an antibody or antibodyfragment as provided herein comprises a heavy chain comprising (i) SEQID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2 and SEQ ID NO: 3 for CDRH3,(ii) SEQ ID NO: 1 for CDRH1, SEQ ID NO: 41 for CDRH2 and SEQ ID NO: 42for CDRH3, (iii) SEQ ID NO: 1 for CDRH1, SEQ ID NO: 41 for CDRH2 and SEQID NO: 43 for CDRH3, or (iv) SEQ ID NO: 17 for CDRH1, SEQ ID NO: 18 forCDRH2 and SEQ ID NO: 19 for CDRH3.

Also provided is an antibody comprising a light chain comprising one ormore (i.e. one, two or all three) light chain CDRs from FI6 variant 1,FI6 variant 2, FI6 variant 3, FI6 variant 4, FI6 variant 5, FI28 variant1 or FI28 variant 2. In an exemplary embodiment, the antibody or antigenbinding fragment of the invention comprises a light chain CDR1 with theamino acid sequence of SEQ ID NO: 4, SEQ ID NO: 44 or SEQ ID NO: 20; alight chain CDR2 with the amino acid sequence of SEQ ID NO: 5 or SEQ IDNO: 21; and a light chain CDR3 with the amino acid sequence of SEQ IDNO: 6 or SEQ ID NO: 22. In certain embodiments, an antibody as providedherein comprises a light chain comprising (i) SEQ ID NO: 4 for CDRL1,SEQ ID NO: 5 for CDRL2 and SEQ ID NO: 6 for CDRL3, (ii) SEQ ID NO: 44for CDRL1, SEQ ID NO: 5 for CDRL2 and SEQ ID NO: 6 for CDRL3 or (iii)SEQ ID NO: 20 for CDRL1, SEQ ID NO: 21 for CDRL2 and SEQ ID NO: 22 forCDRL3.

In one embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody FI6 variant 1 aslisted in Table 2, and neutralizes influenza A virus infection. Inanother embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody FI6 variant 2 aslisted in Table 2, and neutralizes influenza A virus infection. Inanother embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody FI6 variant 3 aslisted in Table 2, and neutralizes influenza A virus infection. Inanother embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody FI6 variant 4 aslisted in Table 2, and neutralizes influenza A virus infection. Inanother embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody FI6 variant 5 aslisted in Table 2, and neutralizes influenza A virus infection.

In yet another embodiment, an antibody of the invention, or antigenbinding fragment thereof, comprises all of the CDRs of antibody FI28variant 1 as listed in Table 2, and neutralizes influenza A virusinfection. In still another embodiment, an antibody of the invention, orantigen binding fragment thereof, comprises all of the CDRs of antibodyFI28 variant 2 as listed in Table 2, and neutralizes influenza A virusinfection.

Examples of antibodies of the invention include, but are not limited to,FI6 variant 1, FI6 variant 2, FI6 variant 3, FI6 variant 4, FI6 variant5, FI28 variant 1 and FI28 variant 2.

The invention further comprises an antibody, or fragment thereof, thatbinds to the same epitope as an antibody of the invention, or anantibody that competes with an antibody or antigen binding fragment ofthe invention.

Antibodies of the invention also include hybrid antibody molecules thatcomprise one or more CDRs from an antibody of the invention and one ormore CDRs from another antibody to the same epitope. In one embodiment,such hybrid antibodies comprise three CDRs from an antibody of theinvention and three CDRs from another antibody to the same epitope.Exemplary hybrid antibodies comprise i) the three light chain CDRs froman antibody of the invention and the three heavy chain CDRs from anotherantibody to the same epitope, or ii) the three heavy chain CDRs from anantibody of the invention and the three light chain CDRs from anotherantibody to the same epitope.

Variant antibodies are also included within the scope of the invention.Thus, variants of the sequences recited in the application are alsoincluded within the scope of the invention. Such variants includenatural variants generated by somatic mutation in vivo during the immuneresponse or in vitro upon culture of immortalized B cell clones.Alternatively, variants may arise due to the degeneracy of the geneticcode, as mentioned above or may be produced due to errors intranscription or translation.

Further variants of the antibody sequences having improved affinityand/or potency may be obtained using methods known in the art and areincluded within the scope of the invention. For example, amino acidsubstitutions may be used to obtain antibodies with further improvedaffinity. Alternatively, codon optimization of the nucleotide sequencemay be used to improve the efficiency of translation in expressionsystems for the production of the antibody. Further, polynucleotidescomprising a sequence optimized for antibody specificity or neutralizingactivity by the application of a directed evolution method to any of thenucleic acid sequences of the invention are also within the scope of theinvention.

In one embodiment variant antibody sequences may share 70% or more (i.e.75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) amino acid sequenceidentity with the sequences recited in the application. In someembodiments such sequence identity is calculated with regard to the fulllength of the reference sequence (i.e. the sequence recited in theapplication). In some further embodiments, percentage identity, asreferred to herein, is as determined using BLAST version 2.1.3 using thedefault parameters specified by the NCBI (the National Center forBiotechnology Information; http://www.ncbi.nlm.nih.gov/) [Blosum 62matrix; gap open penalty=11 and gap extension penalty=1].

In another aspect, the invention also includes nucleic acid sequencesencoding part or all of the light and heavy chains and CDRs of theantibodies of the present invention. Provided herein are nucleic acidsequences encoding part or all of the light and heavy chains and CDRs ofexemplary antibodies of the invention. Table 1 provides the SEQ IDnumbers for the nucleic acid sequences encoding the heavy chain andlight chain variable regions of the exemplary antibodies of theinvention. For example, nucleic acid sequences provided herein includeSEQ ID NO: 15 (encoding the FI6 variant 1 heavy chain variable region),SEQ ID NO: 16 (encoding the FI6 variant 1 and FI6 variant 2 light chainvariable region), SEQ ID NO: 34 (encoding the FI6 variant 2 heavy chainvariable region), SEQ ID NO: 56 (encoding the FI6 variant 3 heavy chainvariable region), SEQ ID NO: 58 (encoding the FI6 variant 3 and FI6variant 4 light chain variable region), SEQ ID NO: 60 (encoding the FI6variant 4 and FI6 variant 5 heavy chain variable region), SEQ ID NO: 62(encoding the FI6 variant 5 light chain variable region), SEQ ID NO: 31(encoding the FI28 variant 1 heavy chain variable region), SEQ ID NO: 36(encoding the FI28 variant 2 heavy chain variable region) and SEQ ID NO:32 (encoding the FI28 variant 1 and variant 2 light chain variableregion).

Table 3 provides the SEQ ID numbers for the nucleic acid sequencesencoding the CDRs of the exemplary antibodies of the invention. Due tothe redundancy of the genetic code, variants of these sequences willexist that encode the same amino acid sequences.

TABLE 3 SEQ ID Numbers for CDR Polynucleotides of Exemplary Influenza AVirus Neutralizing Antibodies: SEQ ID NOs. for CDR Polynucleotides CDRH1CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 FI6 variant 1 7 8 9 10 11 12 FI6 variant 27 8 9 10 11 12 FI6 variant 3 45 46 47 48 49 50 FI6 variant 4 51 52 53 4849 50 FI6 variant 5 51 52 53 54 49 50 FI28 variant 1 23 24 25 26 27 28FI28 variant 2 23 24 25 26 27 28

In one embodiment, nucleic acid sequences according to the inventioninclude nucleic acid sequences having at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% identity to the nucleic acid encoding a heavy or light chainof an antibody of the invention. In another embodiment, a nucleic acidsequence of the invention has the sequence of a nucleic acid encoding aheavy or light chain CDR of an antibody of the invention. For example, anucleic acid sequence according to the invention comprises a sequencethat is at least 75% identical to the nucleic acid sequences of SEQ IDNOs: 7-12, 15, 16, 34, 23-28, 31, 32, 36, 45-54, 56, 58, 60 or 62.

Further included within the scope of the invention are vectors, forexample, expression vectors, comprising a nucleic acid sequenceaccording to the invention. Cells transformed with such vectors are alsoincluded within the scope of the invention. Examples of such cellsinclude but are not limited to, eukaryotic cells, e.g. yeast cells,animal cells or plant cells. In one embodiment the cells are mammalian,e.g. human, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells.

The invention also relates to monoclonal antibodies that bind to anepitope capable of binding the antibodies of the invention, including,but not limited to, a monoclonal antibody selected from the groupconsisting FI6 variant 1, FI6 variant 2, FI6 variant 3, FI6 variant 4,FI6 variant 5, FI28 variant 1 and FI28 variant 2.

Monoclonal and recombinant antibodies are particularly useful inidentification and purification of the individual polypeptides or otherantigens against which they are directed. The antibodies of theinvention have additional utility in that they may be employed asreagents in immunoassays, radioimmunoassays (RIA) or enzyme-linkedimmunosorbent assays (ELISA). In these applications, the antibodies canbe labelled with an analytically-detectable reagent such as aradioisotope, a fluorescent molecule or an enzyme. The antibodies mayalso be used for the molecular identification and characterization(epitope mapping) of antigens.

Antibodies of the invention can be coupled to a drug for delivery to atreatment site or coupled to a detectable label to facilitate imaging ofa site comprising cells of interest, such as cells infected withinfluenza A virus. Methods for coupling antibodies to drugs anddetectable labels are well known in the art, as are methods for imagingusing detectable labels. Labelled antibodies may be employed in a widevariety of assays, employing a wide variety of labels. Detection of theformation of an antibody-antigen complex between an antibody of theinvention and an epitope of interest (an influenza A virus epitope) canbe facilitated by attaching a detectable substance to the antibody.Suitable detection means include the use of labels such asradionuclides, enzymes, coenzymes, fluorescers, chemiluminescers,chromogens, enzyme substrates or co-factors, enzyme inhibitors,prosthetic group complexes, free radicals, particles, dyes, and thelike. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material is luminol; examples of bioluminescentmaterials include luciferase, luciferin, and aequorin; and examples ofsuitable radioactive material include 125I, 131I, 35S, or 3H. Suchlabelled reagents may be used in a variety of well-known assays, such asradioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescentimmunoassays, and the like. (See U.S. Pat. No. 3,766,162; U.S. Pat. No.3,791,932; U.S. Pat. No. 3,817,837; and U.S. Pat. No. 4,233,402 forexample).

An antibody according to the invention may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent, or aradioactive metal ion or radioisotope. Examples of radioisotopesinclude, but are not limited to, I-131, I-123, I-125, Y-90, Re-188,Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and thelike. Such antibody conjugates can be used for modifying a givenbiological response; the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin.

Techniques for conjugating such therapeutic moiety to antibodies arewell known. See, for example, Arnon et al. (1985) “Monoclonal Antibodiesfor Immunotargeting of Drugs in Cancer Therapy,” in MonoclonalAntibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.),pp. 243-256; ed. Hellstrom et al. (1987) “Antibodies for Drug Delivery,”in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker,Inc.), pp. 623-653; Thorpe (1985) “Antibody Carriers of Cytotoxic Agentsin Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biologicaland Clinical Applications, ed. Pinchera et al. pp. 475-506 (EditriceKurtis, Milano, Italy, 1985); “Analysis, Results, and Future Prospectiveof the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy,” inMonoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin etal. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al.(1982) Immunol. Rev. 62:119-158.

Alternatively, an antibody, or antibody fragment thereof, can beconjugated to a second antibody, or antibody fragment thereof, to forman antibody heteroconjugate as described in U.S. Pat. No. 4,676,980. Inaddition, linkers may be used between the labels and the antibodies ofthe invention (e.g. U.S. Pat. No. 4,831,175). Antibodies or,antigen-binding fragments thereof may be directly labelled withradioactive iodine, indium, yttrium, or other radioactive particle knownin the art (e.g. U.S. Pat. No. 5,595,721). Treatment may consist of acombination of treatment with conjugated and non-conjugated antibodiesadministered simultaneously or subsequently (e.g. WO00/52031;WO00/52473).

Antibodies of the invention may also be attached to a solid support.Additionally, antibodies of the invention, or functional antibodyfragments thereof, can be chemically modified by covalent conjugation toa polymer to, for example, increase their circulating half-life.Examples of polymers, and methods to attach them to peptides, are shownin U.S. Pat. No. 4,766,106; U.S. Pat. No. 4,179,337; U.S. Pat. No.4,495,285 and U.S. Pat. No. 4,609,546. In some embodiments the polymersmay be selected from polyoxyethylated polyols and polyethylene glycol(PEG). PEG is soluble in water at room temperature and has the generalformula: R(O—CH2-CH2)n O—R where R can be hydrogen, or a protectivegroup such as an alkyl or alkanol group. In one embodiment theprotective group may have between 1 and 8 carbons. In a furtherembodiment the protective group is methyl. The symbol n is a positiveinteger. In one embodiment n is between 1 and 1,000. In anotherembodiment n is between 2 and 500. In one embodiment the PEG has anaverage molecular weight between 1,000 and 40,000. In a furtherembodiment the PEG has a molecular weight between 2,000 and 20,000. Inyet a further embodiment the PEG has a molecular weight between 3,000and 12,000. In one embodiment PEG has at least one hydroxy group. Inanother embodiment the PEG has a terminal hydroxy group. In yet anotherembodiment it is the terminal hydroxy group which is activated to reactwith a free amino group on the inhibitor. However, it will be understoodthat the type and amount of the reactive groups may be varied to achievea covalently conjugated PEG/antibody of the present invention.

Water-soluble polyoxyethylated polyols are also useful in the presentinvention. They include polyoxyethylated sorbitol, polyoxyethylatedglucose, polyoxyethylated glycerol (POG), and the like. In oneembodiment, POG is used. Without being bound by any theory, because theglycerol backbone of polyoxyethylated glycerol is the same backboneoccurring naturally in, for example, animals and humans in mono-, di-,triglycerides, this branching would not necessarily be seen as a foreignagent in the body. In some embodiments POG has a molecular weight in thesame range as PEG. Another drug delivery system that can be used forincreasing circulatory half-life is the liposome. Methods of preparingliposome delivery systems are discussed in Gabizon et al. (1982), Cafiso(1981) and Szoka (1980). Other drug delivery systems are known in theart and are described in, for example, referenced in Poznansky et al.(1980) and Poznansky (1984).

Antibodies of the invention may be provided in purified form. Typically,the antibody will be present in a composition that is substantially freeof other polypeptides e.g. where less than 90% (by weight), usually lessthan 60% and more usually less than 50% of the composition is made up ofother polypeptides.

Antibodies of the invention may be immunogenic in non-human (orheterologous) hosts e.g. in mice. In particular, the antibodies may havean idiotope that is immunogenic in non-human hosts, but not in a humanhost. Antibodies of the invention for human use include those thatcannot be easily isolated from hosts such as mice, goats, rabbits, rats,non-primate mammals, etc. and cannot generally be obtained byhumanisation or from xeno-mice.

Antibodies of the invention can be of any isotype (e.g. IgA, IgG, IgMi.e. an α, γ or μ heavy chain), but will generally be IgG. Within theIgG isotype, antibodies may be IgG1, IgG2, IgG3 or IgG4 subclass.Antibodies of the invention may have a κ or a λ light chain.

Production of Antibodies

Antibodies according to the invention can be made by any method known inthe art. For example, the general methodology for making monoclonalantibodies using hybridoma technology is well known (Kohler, G. andMilstein, C. 1975; Kozbar et al. 1983). In one embodiment, thealternative EBV immortalisation method described in WO2004/076677 isused.

Using the method described in WO2004/076677, B cells producing theantibody of the invention can be transformed with EBV in the presence ofa polyclonal B cell activator. Transformation with EBV is a standardtechnique and can easily be adapted to include polyclonal B cellactivators.

Additional stimulants of cellular growth and differentiation mayoptionally be added during the transformation step to further enhancethe efficiency. These stimulants may be cytokines such as IL-2 andIL-15. In one aspect, IL-2 is added during the immortalisation step tofurther improve the efficiency of immortalisation, but its use is notessential. The immortalized B cells produced using these methods canthen be cultured using methods known in the art and antibodies isolatedtherefrom.

Using the method described in UK Patent Application 0819376.5, singleplasma cells can be cultured in microwell culture plates. Antibodies canbe isolated from the single plasma cell cultures. Further, from singleplasma cell cultures, RNA can be extracted and single cell PCR can beperformed using methods known in the art. The VH and VL regions of theantibodies can be amplified by RT-PCR, sequenced and cloned into anexpression vector that is then transfected into HEK293T cells or otherhost cells. The cloning of nucleic acid in expression vectors, thetransfection of host cells, the culture of the transfected host cellsand the isolation of the produced antibody can be done using any methodsknown to one of skill in the art.

The antibodies may be further purified, if desired, using filtration,centrifugation and various chromatographic methods such as HPLC oraffinity chromatography. Techniques for purification of antibodies,e.g., monoclonal antibodies, including techniques for producingpharmaceutical-grade antibodies, are well known in the art.

Fragments of the antibodies of the invention can be obtained from theantibodies by methods that include digestion with enzymes, such aspepsin or papain, and/or by cleavage of disulfide bonds by chemicalreduction. Alternatively, fragments of the antibodies can be obtained bycloning and expression of part of the sequences of the heavy or lightchains. Antibody “fragments” may include Fab, Fab′, F(ab′)2 and Fvfragments. The invention also encompasses single-chain Fv fragments(scFv) derived from the heavy and light chains of an antibody of theinvention e.g. the invention includes a scFv comprising the CDRs from anantibody of the invention. Also included are heavy or light chainmonomers and dimers, single domain heavy chain antibodies, single domainlight chain antibodies, as well as single chain antibodies, e.g. singlechain Fv in which the heavy and light chain variable domains are joinedby a peptide linker.

Antibody fragments of the invention may impart monovalent or multivalentinteractions and be contained in a variety of structures as describedabove. For instance, scFv molecules may be synthesized to create atrivalent “triabody” or a tetravalent “tetrabody.” The scFv moleculesmay include a domain of the Fc region resulting in bivalent minibodies.In addition, the sequences of the invention may be a component ofmultispecific molecules in which the sequences of the invention targetthe epitopes of the invention and other regions of the molecule bind toother targets. Exemplary molecules include, but are not limited to,bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies(Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

Standard techniques of molecular biology may be used to prepare DNAsequences encoding the antibodies or antibody fragments of the presentinvention. Desired DNA sequences may be synthesised completely or inpart using oligonucleotide synthesis techniques. Site-directedmutagenesis and polymerase chain reaction (PCR) techniques may be usedas appropriate.

Any suitable host cell/vector system may be used for expression of theDNA sequences encoding the antibody molecules of the present inventionor fragments thereof. Bacterial, for example E. coli, and othermicrobial systems may be used, in part, for expression of antibodyfragments such as Fab and F(ab′)2 fragments, and especially Fv fragmentsand single chain antibody fragments, for example, single chain Fvs.Eukaryotic, e.g. mammalian, host cell expression systems may be used forproduction of larger antibody molecules, including complete antibodymolecules. Suitable mammalian host cells include, but are not limitedto, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells.

The present invention also provides a process for the production of anantibody molecule according to the present invention comprisingculturing a host cell comprising a vector encoding a nucleic acid of thepresent invention under conditions suitable for leading to expression ofprotein from DNA encoding the antibody molecule of the presentinvention, and isolating the antibody molecule.

The antibody molecule may comprise only a heavy or light chainpolypeptide, in which case only a heavy chain or light chain polypeptidecoding sequence needs to be used to transfect the host cells. Forproduction of products comprising both heavy and light chains, the cellline may be transfected with two vectors, a first vector encoding alight chain polypeptide and a second vector encoding a heavy chainpolypeptide. Alternatively, a single vector may be used, the vectorincluding sequences encoding light chain and heavy chain polypeptides.

Alternatively, antibodies according to the invention may be produced byi) expressing a nucleic acid sequence according to the invention in ahost cell, and ii) isolating the expressed antibody product.Additionally, the method may include iii) purifying the antibody.

Screening of Transformed B Cells, Cultured Single Plasma Cells andTransfected HEK293T Cells

Transformed B cells and cultured single plasma cells may be screened forthose producing antibodies of the desired specificity or function.

The screening step may be carried out by any immunoassay, for example,ELISA, by staining of tissues or cells (including transfected cells), byneutralization assay or by one of a number of other methods known in theart for identifying desired specificity or function. The assay mayselect on the basis of simple recognition of one or more antigens, ormay select on the additional basis of a desired function e.g. to selectneutralizing antibodies rather than just antigen-binding antibodies, toselect antibodies that can change characteristics of targeted cells,such as their signalling cascades, their shape, their growth rate, theircapability of influencing other cells, their response to the influenceby other cells or by other reagents or by a change in conditions, theirdifferentiation status, etc.

Individual transformed B cell clones may then be produced from thepositive transformed B cell culture. The cloning step for separatingindividual clones from the mixture of positive cells may be carried outusing limiting dilution, micromanipulation, single cell deposition bycell sorting or another method known in the art.

Nucleic acid from the cultured single plasma cells can be isolated,cloned and expressed in HEK293T cells or other host cells using methodsknown in the art.

The immortalized B cell clones or the transfected HEK293T cells of theinvention can be used in various ways e.g. as a source of monoclonalantibodies, as a source of nucleic acid (DNA or mRNA) encoding amonoclonal antibody of interest, for research, etc.

The invention provides a composition comprising immortalized B memorycells or transfected host cells that produce antibodies that neutralizeat least two different subtypes of influenza A virus selected from group1 and group 2 subtypes.

Epitopes

As mentioned above, the antibodies of the invention can be used to mapthe epitopes to which they bind. The inventors have discovered that theantibodies neutralizing influenza A virus infection are directed towardsepitopes found on HA. In one embodiment, the antibodies are directed toone or more epitopes in the stem region of HA that are conserved amongone or more group 1 and group 2 subtypes of influenza A virus. Theepitopes to which the antibodies of the invention bind may be linear(continuous) or conformational (discontinuous). In one embodiment, theantibodies and antibody fragments of the invention bind a region of thepolypeptide comprising SEQ ID NOs: 37, 38, 39 or 40, as discussedherein.

In another embodiment, the epitope to which the antibodies of theinvention bind comprises amino acid residues in the HA1 and HA2polypeptides of one or two HA monomers as described above. The HAmonomers can be uncleaved or cleaved.

The epitopes recognized by the antibodies of the present invention mayhave a number of uses. The epitope and mimotopes thereof in purified orsynthetic form can be used to raise immune responses (i.e., as avaccine, or for the production of antibodies for other uses) or forscreening sera for antibodies that immunoreact with the epitope ormimotopes thereof. In one embodiment such an epitope or mimotope, orantigen comprising such an epitope or mimotope may be used as a vaccinefor raising an immune response. The antibodies and antibody fragments ofthe invention can also be used in a method of monitoring the quality ofvaccines. In particular the antibodies can be used to check that theantigen in a vaccine contains the correct immunogenic epitope in thecorrect conformation.

The epitope may also be useful in screening for ligands that bind tosaid epitope. Such ligands, include but are not limited to antibodies;including those from camels, sharks and other species, fragments ofantibodies, peptides, phage display technology products, aptamers,adnectins or fragments of other viral or cellular proteins, may blockthe epitope and so prevent infection. Such ligands are encompassedwithin the scope of the invention.

Recombinant Expression

The immortalized B cell clone or the cultured plasma cell of theinvention may also be used as a source of nucleic acid for the cloningof antibody genes for subsequent recombinant expression. Expression fromrecombinant sources is more common for pharmaceutical purposes thanexpression from B cells or hybridomas e.g. for reasons of stability,reproducibility, culture ease, etc.

Thus the invention provides a method for preparing a recombinant cell,comprising the steps of: (i) obtaining one or more nucleic acids (e.g.heavy and/or light chain mRNAs) from the B cell clone or the culturedsingle plasma cell that encodes the antibody of interest; (ii) insertingthe nucleic acid into an expression vector and (iii) transfecting thevector into a host cell in order to permit expression of the antibody ofinterest in that host cell.

Similarly, the invention provides a method for preparing a recombinantcell, comprising the steps of: (i) sequencing nucleic acid(s) from the Bcell clone or the cultured single plasma cell that encodes the antibodyof interest; and (ii) using the sequence information from step (i) toprepare nucleic acid(s) for insertion into a host cell in order topermit expression of the antibody of interest in that host cell. Thenucleic acid may, but need not, be manipulated between steps (i) and(ii) to introduce restriction sites, to change codon usage, and/or tooptimise transcription and/or translation regulatory sequences.

The invention also provides a method of preparing a transfected hostcell, comprising the step of transfecting a host cell with one or morenucleic acids that encode an antibody of interest, wherein the nucleicacids are nucleic acids that were derived from an immortalized B cellclone or a cultured single plasma cell of the invention. Thus theprocedures for first preparing the nucleic acid(s) and then using it totransfect a host cell can be performed at different times by differentpeople in different places (e.g. in different countries).

These recombinant cells of the invention can then be used for expressionand culture purposes. They are particularly useful for expression ofantibodies for large-scale pharmaceutical production. They can also beused as the active ingredient of a pharmaceutical composition. Anysuitable culture technique can be used, including but not limited tostatic culture, roller bottle culture, ascites fluid, hollow-fiber typebioreactor cartridge, modular minifermenter, stirred tank, microcarrierculture, ceramic core perfusion, etc.

Methods for obtaining and sequencing immunoglobulin genes from B cellsor plasma cells are well known in the art (e.g. see Chapter 4 of KubyImmunology, 4th edition, 2000).

The transfected host cell may be a eukaryotic cell, including yeast andanimal cells, particularly mammalian cells (e.g. CHO cells, NS0 cells,human cells such as PER.C6 (Jones et al 2003) or HKB-11 (Cho et al.2001; Cho et al. 2003) cells, myeloma cells (U.S. Pat. No. 5,807,715;U.S. Pat. No. 6,300,104 etc.)), as well as plant cells. Preferredexpression hosts can glycosylate the antibody of the invention,particularly with carbohydrate structures that are not themselvesimmunogenic in humans. In one embodiment the transfected host cell maybe able to grow in serum-free media. In a further embodiment thetransfected host cell may be able to grow in culture without thepresence of animal-derived products. The transfected host cell may alsobe cultured to give a cell line.

The invention provides a method for preparing one or more nucleic acidmolecules (e.g. heavy and light chain genes) that encode an antibody ofinterest, comprising the steps of: (i) preparing an immortalized B cellclone or culturing a plasma cell according to the invention; (ii)obtaining from the B cell clone or the cultured single plasma cellnucleic acid that encodes the antibody of interest. The invention alsoprovides a method for obtaining a nucleic acid sequence that encodes anantibody of interest, comprising the steps of: (i) preparing animmortalized B cell clone or culturing a single plasma cell according tothe invention; (ii) sequencing nucleic acid from the B cell clone or thecultured plasma cell that encodes the antibody of interest.

The invention also provides a method of preparing nucleic acidmolecule(s) that encodes an antibody of interest, comprising the step ofobtaining the nucleic acid that was obtained from a transformed B cellclone or a cultured plasma cell of the invention. Thus the proceduresfor first obtaining the B cell clone or the cultured plasma cell, andthen obtaining nucleic acid(s) from the B cell clone or the culturedplasma cell can be performed at different times by different people indifferent places (e.g. in different countries).

The invention provides a method for preparing an antibody (e.g. forpharmaceutical use), comprising the steps of: (i) obtaining and/orsequencing one or more nucleic acids (e.g. heavy and light chain genes)from the selected B cell clone or the cultured plasma cell expressingthe antibody of interest; (ii) inserting the nucleic acid(s) into orusing the nucleic acid(s) sequence(s) to prepare an expression vector;(iii) transfect a host cell that can express the antibody of interest;(iv) culturing or sub-culturing the transfected host cells underconditions where the antibody of interest is expressed; and, optionally,(v) purifying the antibody of interest.

The invention also provides a method of preparing an antibody comprisingthe steps of: culturing or sub-culturing a transfected host cellpopulation under conditions where the antibody of interest is expressedand, optionally, purifying the antibody of interest, wherein saidtransfected host cell population has been prepared by (i) providingnucleic acid(s) encoding a selected antibody of interest that isproduced by a B cell clone or a cultured plasma cell prepared asdescribed above, (ii) inserting the nucleic acid(s) into an expressionvector, (iii) transfecting the vector in a host cell that can expressthe antibody of interest, and (iv) culturing or sub-culturing thetransfected host cell comprising the inserted nucleic acids to producethe antibody of interest. Thus the procedures for first preparing therecombinant host cell and then culturing it to express antibody can beperformed at very different times by different people in differentplaces (e.g., in different countries).

Pharmaceutical Compositions

The invention provides a pharmaceutical composition containing theantibodies and/or antibody fragments of the invention and/or nucleicacid encoding such antibodies and/or the epitopes recognised by theantibodies of the invention. A pharmaceutical composition may alsocontain a pharmaceutically acceptable carrier to allow administration.The carrier should not itself induce the production of antibodiesharmful to the individual receiving the composition and should not betoxic. Suitable carriers may be large, slowly metabolised macromoleculessuch as proteins, polypeptides, liposomes, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymersand inactive virus particles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the subject.

Within the scope of the invention, forms of administration may includethose forms suitable for parenteral administration, e.g. by injection orinfusion, for example by bolus injection or continuous infusion. Wherethe product is for injection or infusion, it may take the form of asuspension, solution or emulsion in an oily or aqueous vehicle and itmay contain formulatory agents, such as suspending, preservative,stabilising and/or dispersing agents. Alternatively, the antibodymolecule may be in dry form, for reconstitution before use with anappropriate sterile liquid.

Once formulated, the compositions of the invention can be administereddirectly to the subject. In one embodiment the compositions are adaptedfor administration to human subjects.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intraperitoneal,intrathecal, intraventricular, transdermal, transcutaneous, topical,subcutaneous, intranasal, enteral, sublingual, intravaginal or rectalroutes. Hyposprays may also be used to administer the pharmaceuticalcompositions of the invention. Typically, the therapeutic compositionsmay be prepared as injectables, either as liquid solutions orsuspensions. Solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection may also be prepared.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Dosage treatmentmay be a single dose schedule or a multiple dose schedule. Knownantibody-based pharmaceuticals provide guidance relating to frequency ofadministration e.g. whether a pharmaceutical should be delivered daily,weekly, monthly, etc. Frequency and dosage may also depend on theseverity of symptoms.

Compositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. Solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared (e.g. a lyophilised composition, like Synagis™ and Herceptin™,for reconstitution with sterile water containing a preservative). Thecomposition may be prepared for topical administration e.g. as anointment, cream or powder. The composition may be prepared for oraladministration e.g. as a tablet or capsule, as a spray, or as a syrup(optionally flavoured). The composition may be prepared for pulmonaryadministration e.g. as an inhaler, using a fine powder or a spray. Thecomposition may be prepared as a suppository or pessary. The compositionmay be prepared for nasal, aural or ocular administration e.g. as drops.The composition may be in kit form, designed such that a combinedcomposition is reconstituted just prior to administration to a subject.For example, a lyophilised antibody can be provided in kit form withsterile water or a sterile buffer.

It will be appreciated that the active ingredient in the compositionwill be an antibody molecule, an antibody fragment or variants andderivatives thereof. As such, it will be susceptible to degradation inthe gastrointestinal tract. Thus, if the composition is to beadministered by a route using the gastrointestinal tract, thecomposition will need to contain agents which protect the antibody fromdegradation but which release the antibody once it has been absorbedfrom the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Gennaro (2000) Remington: The Science and Practice ofPharmacy, 20th edition, ISBN: 0683306472.

Pharmaceutical compositions of the invention generally have a pH between5.5 and 8.5, in some embodiments this may be between 6 and 8, and infurther embodiments about 7. The pH may be maintained by the use of abuffer. The composition may be sterile and/or pyrogen free. Thecomposition may be isotonic with respect to humans. In one embodimentpharmaceutical compositions of the invention are supplied inhermetically-sealed containers.

Pharmaceutical compositions will include an effective amount of one ormore antibodies of the invention and/or a polypeptide comprising anepitope that binds an antibody of the invention i.e. an amount that issufficient to treat, ameliorate, or prevent a desired disease orcondition, or to exhibit a detectable therapeutic effect. Therapeuticeffects also include reduction in physical symptoms. The preciseeffective amount for any particular subject will depend upon their sizeand health, the nature and extent of the condition, and the therapeuticsor combination of therapeutics selected for administration. Theeffective amount for a given situation is determined by routineexperimentation and is within the judgment of a clinician. For purposesof the present invention, an effective dose will generally be from about0.01 mg/kg to about 50 mg/kg, or about 0.05 mg/kg to about 10 mg/kg ofthe compositions of the present invention in the individual to which itis administered. Known antibody-based pharmaceuticals provide guidancein this respect e.g. Herceptin™ is administered by intravenous infusionof a 21 mg/ml solution, with an initial loading dose of 4 mg/kg bodyweight and a weekly maintenance dose of 2 mg/kg body weight; Rituxan™ isadministered weekly at 375 mg/m2; etc.

In one embodiment compositions can include more than one (e.g. 2, 3,etc.) antibodies of the invention to provide an additive or synergistictherapeutic effect. In another embodiment, the composition may compriseone or more (e.g. 2, 3, etc.) antibodies of the invention and one ormore (e.g. 2, 3, etc.) additional antibodies against influenza A orinfluenza B virus. For example, one antibody may bind to a HA epitope,while another may bind to a different epitope on HA, or to an epitope onthe neuraminidase and/or matrix proteins. Further, the administration ofantibodies of the invention together with an influenza A vaccine or withantibodies of specificities other than influenza A virus, for example,influenza B virus, are within the scope of the invention. The antibodiesof the invention can be administered either combined/simultaneously orat separate times from an influenza vaccine or from antibodies ofspecificities other than influenza A virus.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising two or more antibodies, wherein the firstantibody is an antibody of the invention and is specific for an HAepitope, and the second antibody is specific for a neuraminidaseepitope, a second HA epitope and/or a matrix epitope. For example, theinvention provides a pharmaceutical composition comprising two or moreantibodies, wherein the first antibody is specific for an epitope in thestem of an influenza A virus HA, and the second antibody is specific fora neuraminidase epitope, a second HA epitope (for example, an epitope inthe globular head of HA, a second epitope in the stem of HA), and/or amatrix epitope. The second epitope in the stem or the epitope in theglobular head of the influenza A virus HA may, but need not, beconserved among more than one influenza A virus subtype.

In yet another embodiment, the invention provides a pharmaceuticalcomposition comprising two or more antibodies, wherein the firstantibody is specific for a neuraminidase epitope, and the secondantibody is specific for a second neuraminidase epitope, a HA epitopeand/or a matrix epitope.

In still another embodiment, the invention provides a pharmaceuticalcomposition comprising two or more antibodies, wherein the firstantibody is specific for a matrix epitope, and the second antibody isspecific for a second matrix epitope, an epitope on HA and/orneuraminidase.

Exemplary antibodies of the invention specific for an Influenza A virustarget protein include, but are not limited to, FI6 variant 1, FI6variant 2, FI6 variant 3, FI6 variant 4, FI6 variant 5, FI28 variant 1or FI28 variant 2.

In one embodiment, the invention provides a pharmaceutical compositioncomprising the antibody FI6 variant 1 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody FI6 variant 2 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody FI6 variant 3 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody FI6 variant 4 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody FI6 variant 5 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In yet anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody FI28 variant 1 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In still anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody FI28 variant 2 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier.

Antibodies of the invention may be administered (either combined orseparately) with other therapeutics e.g. with chemotherapeuticcompounds, with radiotherapy, etc. In one embodiment, the therapeuticcompounds include anti-viral compounds such as Tamiflu™. Suchcombination therapy provides an additive or synergistic improvement intherapeutic efficacy relative to the individual therapeutic agents whenadministered alone. The term “synergy” is used to describe a combinedeffect of two or more active agents that is greater than the sum of theindividual effects of each respective active agent. Thus, where thecombined effect of two or more agents results in “synergisticinhibition” of an activity or process, it is intended that theinhibition of the activity or process is greater than the sum of theinhibitory effects of each respective active agent. The term“synergistic therapeutic effect” refers to a therapeutic effect observedwith a combination of two or more therapies wherein the therapeuticeffect (as measured by any of a number of parameters) is greater thanthe sum of the individual therapeutic effects observed with therespective individual therapies.

Antibodies may be administered to those subjects who have previouslyshown no response to treatment for influenza A virus infection, i.e.have been shown to be refractive to anti-influenza treatment. Suchtreatment may include previous treatment with an anti-viral agent. Thismay be due to, for example, infection with an anti-viral resistantstrain of influenza A virus.

In one embodiment, a composition of the invention may include antibodiesof the invention, wherein the antibodies may make up at least 50% byweight (e.g. 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more)of the total protein in the composition. In such a composition, theantibodies are in purified form.

The invention provides a method of preparing a pharmaceutical,comprising the steps of: (i) preparing an antibody of the invention; and(ii) admixing the purified antibody with one or morepharmaceutically-acceptable carriers.

The invention also provides a method of preparing a pharmaceutical,comprising the step of admixing an antibody with one or morepharmaceutically-acceptable carriers, wherein the antibody is amonoclonal antibody that was obtained from a transformed B cell or acultured plasma cell of the invention. Thus the procedures for firstobtaining the monoclonal antibody and then preparing the pharmaceuticalcan be performed at very different times by different people indifferent places (e.g. in different countries).

As an alternative to delivering antibodies or B cells for therapeuticpurposes, it is possible to deliver nucleic acid (typically DNA) thatencodes the monoclonal antibody (or active fragment thereof) of interestderived from the B cell or the cultured plasma cell to a subject, suchthat the nucleic acid can be expressed in the subject in situ to providea desired therapeutic effect. Suitable gene therapy and nucleic aciddelivery vectors are known in the art.

Compositions of the invention may be immunogenic compositions, and insome embodiments may be vaccine compositions comprising an antigencomprising an epitope recognized by an antibody of the invention.Vaccines according to the invention may either be prophylactic (i.e. toprevent infection) or therapeutic (i.e. to treat infection). In oneembodiment, the invention provides a vaccine comprising a polypeptidecomprising the amino acid sequence of SEQ ID NOs: 37, 38, 39 or 40. Inanother embodiment, the invention provides a vaccine comprising apolypeptide comprising the amino acid residues in the HA1 and HA2polypeptides of one or two HA monomers as described above. The HAmonomers can be uncleaved or cleaved.

Compositions may include an antimicrobial, particularly if packaged in amultiple dose format. They may comprise detergent e.g. a Tween(polysorbate), such as Tween 80. Detergents are generally present at lowlevels e.g. <0.01%. Compositions may also include sodium salts (e.g.sodium chloride) to give tonicity. A concentration of 10+2 mg/ml NaCl istypical.

Further, compositions may comprise a sugar alcohol (e.g. mannitol) or adisaccharide (e.g. sucrose or trehalose) e.g., at around 15-30 mg/ml(e.g. 25 mg/ml), particularly if they are to be lyophilised or if theyinclude material which has been reconstituted from lyophilised material.The pH of a composition for lyophilisation may be adjusted to around 6.1prior to lyophilisation.

The compositions of the invention may also comprise one or moreimmunoregulatory agents. In one embodiment, one or more of theimmunoregulatory agents include(s) an adjuvant.

The epitope compositions of the invention may elicit both a cellmediated immune response as well as a humoral immune response in orderto effectively address influenza A virus infection. This immune responsemay induce long lasting (e.g., neutralizing) antibodies and a cellmediated immunity that can quickly respond upon exposure to influenza Avirus.

Medical Treatments and Uses

The antibodies and antibody fragments of the invention or derivativesand variants thereof may be used for the treatment of influenza A virusinfection, for the prevention of influenza A virus infection or for thediagnosis of influenza A virus infection.

Methods of diagnosis may include contacting an antibody or an antibodyfragment with a sample. Such samples may be tissue samples taken from,for example, nasal passages, sinus cavities, salivary glands, lung,liver, pancreas, kidney, ear, eye, placenta, alimentary tract, heart,ovaries, pituitary, adrenals, thyroid, brain or skin. The methods ofdiagnosis may also include the detection of an antigen/antibody complex.

The invention therefore provides (i) an antibody, an antibody fragment,or variants and derivatives thereof according to the invention, (ii) animmortalized B cell clone according to the invention, (iii) an epitopecapable of binding an antibody of the invention or (iv) a ligand,preferably an antibody, capable of binding an epitope that binds anantibody of the invention for use in therapy.

The invention also provides a method of treating a subject comprisingadministering to the subject an antibody, an antibody fragment, orvariants and derivatives thereof according to the invention, or, aligand, preferably an antibody, capable of binding an epitope that bindsan antibody of the invention. In one embodiment, the method results inreduced influenza A virus infection in the subject. In anotherembodiment, the method prevents, reduces the risk or delays influenza Avirus infection in the subject.

The invention also provides the use of (i) an antibody, an antibodyfragment, or variants and derivatives thereof according to theinvention, (ii) an immortalized B cell clone according to the invention,(iii) an epitope capable of binding an antibody of the invention, or(iv) a ligand, preferably an antibody, that binds to an epitope capableof binding an antibody of the invention, in the manufacture of amedicament for the prevention or treatment of influenza A virusinfection.

The invention provides a composition of the invention for use as amedicament for the prevention or treatment of a influenza A virusinfection. It also provides the use of an antibody of the inventionand/or a protein comprising an epitope to which such an antibody bindsin the manufacture of a medicament for treatment of a subject and/ordiagnosis in a subject. It also provides a method for treating asubject, comprising the step of administering to the subject acomposition of the invention. In some embodiments the subject may be ahuman. One way of checking efficacy of therapeutic treatment involvesmonitoring disease symptoms after administration of the composition ofthe invention. Treatment can be a single dose schedule or a multipledose schedule.

In one embodiment, an antibody, antibody fragment, immortalized B cellclone, epitope or composition according to the invention is administeredto a subject in need of such treatment. Such a subject includes, but isnot limited to, one who is particularly at risk of or susceptible toinfluenza A virus infection, including, for example, animmunocompromised subject. The antibody or antibody fragment of theinvention can also be used in passive immunisation or activevaccination.

Antibodies and fragments thereof as described in the present inventionmay also be used in a kit for the diagnosis of influenza A virusinfection. Further, epitopes capable of binding an antibody of theinvention may be used in a kit for monitoring the efficacy ofvaccination procedures by detecting the presence of protectiveanti-influenza A virus antibodies. Antibodies, antibody fragment, orvariants and derivatives thereof, as described in the present inventionmay also be used in a kit for monitoring vaccine manufacture with thedesired immunogenicity.

The invention also provides a method of preparing a pharmaceutical,comprising the step of admixing a monoclonal antibody with one or morepharmaceutically-acceptable carriers, wherein the monoclonal antibody isa monoclonal antibody that was obtained from a transfected host cell ofthe invention. Thus the procedures for first obtaining the monoclonalantibody (e.g. expressing it and/or purifying it) and then admixing itwith the pharmaceutical carrier(s) can be performed at very differenttimes by different people in different places (e.g. in differentcountries).

Starting with a transformed B cell or a cultured plasma cell of theinvention, various steps of culturing, sub-culturing, cloning,sub-cloning, sequencing, nucleic acid preparation etc. can be performedin order to perpetuate the antibody expressed by the transformed B cellor the cultured plasma cell, with optional optimization at each step. Ina preferred embodiment, the above methods further comprise techniques ofoptimization (e.g. affinity maturation or optimization) applied to thenucleic acids encoding the antibody. The invention encompasses allcells, nucleic acids, vectors, sequences, antibodies etc. used andprepared during such steps.

In all these methods, the nucleic acid used in the expression host maybe manipulated to insert, delete or amend certain nucleic acidsequences. Changes from such manipulation include, but are not limitedto, changes to introduce restriction sites, to amend codon usage, to addor optimise transcription and/or translation regulatory sequences, etc.It is also possible to change the nucleic acid to alter the encodedamino acids. For example, it may be useful to introduce one or more(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions,deletions and/or insertions into the antibody's amino acid sequence.Such point mutations can modify effector functions, antigen-bindingaffinity, post-translational modifications, immunogenicity, etc., canintroduce amino acids for the attachment of covalent groups (e.g.labels) or can introduce tags (e.g. for purification purposes).Mutations can be introduced in specific sites or can be introduced atrandom, followed by selection (e.g. molecular evolution). For instance,one or more nucleic acids encoding any of the CDR regions, heavy chainvariable regions or light chain variable regions of antibodies of theinvention can be randomly or directionally mutated to introducedifferent properties in the encoded amino acids. Such changes can be theresult of an iterative process wherein initial changes are retained andnew changes at other nucleotide positions are introduced. Moreover,changes achieved in independent steps may be combined. Differentproperties introduced into the encoded amino acids may include, but arenot limited to, enhanced affinity.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x means, for example,x+10%.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder” and“condition” (as in medical condition), in that all reflect an abnormalcondition of the human or animal body or of one of its parts thatimpairs normal functioning, is typically manifested by distinguishingsigns and symptoms, and causes the human or animal to have a reducedduration or quality of life.

As used herein, reference to “treatment” of a subject or patient isintended to include prevention, prophylaxis and therapy. The terms“subject” or “patient” are used interchangeably herein to mean allmammals including humans. Examples of subjects include humans, cows,dogs, cats, horses, goats, sheep, pigs, and rabbits. In one embodiment,the patient is a human.

EXAMPLES

Exemplary embodiments of the present invention are provided in thefollowing examples. The following examples are presented only by way ofillustration and to assist one of ordinary skill in using the invention.The examples are not intended in any way to otherwise limit the scope ofthe invention.

Example 1 Generation and Characterization of Influenza A Virus BroadlyNeutralizing Antibodies from Plasma Cells

To identify individuals that may produce heterosubtypic antibodies inresponses to the seasonal influenza vaccine (containing H1 and H3 HAs),we screened by ELISPOT circulating plasma cells collected on day 7 afterboost for their capacity to secrete antibodies that bound to vaccine orto an unrelated H5 HA (A/VN/1203/04). Strikingly, while in four of thefive donors tested H5-specific plasma cells were undetectable, in onedonor 14% of IgG-secreting plasma cells produced antibodies to H5, while57% produced antibodies to the vaccine. CD138+plasma cells were isolatedfrom peripheral blood mononuclear cells (PBMCs) collected 7 days aftervaccination using magnetic micro-beads followed by cell-sorting using aFACSAria machine. Limiting numbers of plasma cells were seeded inmicrowell culture plates. The culture supernatants were tested in threeparallel ELISAs using as antigens recombinant H5 or H9 HAs and theirrelevant antigen tetanus toxoid. Out of the 4,928 culture supernatantsscreened, 12 bound to H5 but not H9 HA, 25 to H9 but not H5 HA and 54 toboth H5 and H9. Some of the 54 cultures with highest OD signal weresubjected to RT-PCR and two paired VH and VL genes were retrieved.

The VH and VL genes were cloned into expression vectors and recombinantantibodies were produced by transfecting HEK293T cells. The twomonoclonal antibodies, FI6 variant 2 and FI28, shared most V, D and Jgene fragments (IGHV3-30*01, IGHD3-9*01, IGHJ4*02 and IGKV4-1*01), butdiffered in the N regions, in the IGKJ usage and in the pattern ofsomatic mutations and were therefore not clonally related.

The specificity of recombinant antibodies was investigated by ELISAusing a panel of HAs belonging to different subtypes. Remarkably, FI6bound all influenza A HA subtypes tested including group 1 (H1, H5 andH9) and group 2 (H3 and H7), while it did not bind HA from influenza Bvirus. In contrast FI28 bound only to the three group 1 HA (H1, H5 andH9).

TABLE 4 Binding to HA by ELISA (% of subtype specific controlantibodies) H1 H3 H5 H7 H9 A/NC/ A/BR/ A/VN/ A/NL/ A/HK/ 20/99 10/071203/04 219/03 1073/99 FI6 variant 2 85.9 68.5 73.7 87.9 98.7 FI28variant 1 59.4 1.3 46.3 −0.5 87.7

Given the homology of VH and VL sequences of the two antibodies,shuffling experiments were performed using H and L chains of FI6 variant2, FI28 variant 1, and 7I13, a hCMV-specific antibody that uses the sameV, D and J elements of the H chain. While binding to H7 required thepairing of FI6 variant 2 H and L chains, binding to H5 was maintainedwhen the FI6 variant 2 and FI28 variant 1 L chains were shuffled. Inaddition H5 binding was also observed when the H chain of FI6 variant 2was paired to the unrelated 7113 L chain. In contrast H5 binding was notobserved when the homologous 7113 H chain was paired with FI6 variant 2L chain. Without being bound by any particular theory, these resultssuggest that the main contribution to H5 binding is from the H chain,while H7 binding requires a precise pairing between H and L chains ofFI6 variant 2.

FI6 variant 2 and FI28 variant 1 were then tested for their capacity toneutralize group 1 and group 2 influenza A subtypes using pseudotypedviruses (Table 5) as well as infectious viruses (Table 6). RemarkablyFI6 variant 2 neutralized all pseudoviruses tested, including six H5isolates belonging to the antigenically divergent clades 0, 1, 2.1, 2.2and 2.3, and two H7 avian isolates. In addition FI6 variant 2neutralized all infectious viruses tested, including two H3N2 virusesand four H1N1 viruses spanning several decades, up to the recent H1N1pandemic isolate A/CA/04/09 (Table 6). FI28 variant 1 neutralized all H5pseudoviruses but did not neutralize H7 pseudoviruses as well as all theinfectious viruses tested. The neutralizing titers on pseudoviruses werehigher than titers on infectious viruses.

TABLE 5 Neutralization of HA-pseudotypes (IC90, μg/ml) H5N1 H7N1 A/HK/A/HK/ A/VN/ A/INDO/ A/WS/ A/AH/ A/ck/IT/ A/ck/FPV/ 491/97 213/03 1203/045/05 MONG/05 1/05 13474/99 Ro/34 FI6 variant 2 0.07 0.02 0.02 0.31 0.030.05 1.87 0.09 FI28 variant 1 0.05 0.33 0.02 0.35 0.04 0.05 >100 >100

TABLE 6 Neutralization of infectious viruses (IC50, μg/ml) H1N1 H3N2A/PR/ A/NC/ A/SI/ A/CA/ A/CA/ A/WI/ 8/34 20/99 3/06 4/09 7/04 67/05 FI6variant 2 2.2 6.3 8.8 12.5 7.9 12.5 FI28 variant 1 >100 >100 >100nd >100 >100 nd, not done

Example 2 FI6 Variant 2 and FI28 Variant 1 Antigenic Binding Sites

To identify the antigenic sites to which the antibodies FI6 variant 2and FI28 variant 1 bind, we first tested their capacity to inhibitbinding of C179, a mouse monoclonal antibody that was mapped to aconserved region of the HA stem region (Y. Okuno, et al., J Virol 67,2552 (1993)). Both FI6 variant 2 and FI28 variant 1 completely inhibitedbinding of C179 to recombinant H5 VN/1203/04 HA, indicating that theyrecognize an overlapping epitope. In contrast, FI6 variant 2 and FI28variant 1 did not compete with a panel of H5-specific antibodiesisolated from H5N1 immune donors that recognize different epitopes inthe globular head of the HA (C. P. Simmons et al., PLoS Med 4, e178(2007); S. Khurana et al., PLoS Med 6, e1000049 (2009)). Attempts to mapthe FI6 variant 2 epitope by selection of escape mutants failed,suggesting that its epitope cannot be easily mutated withoutcompromising viral fitness.

We next performed peptide-based mapping using libraries of linear andcyclised peptides of HA A/VN/1194/04 as well as helix-scan using thesystems of Pepscan Presto BV (Lelystad, The Netherlands). This analysisidentified a binding region of FI6 variant 2 that includes the HA2fusion peptide FGAIAG (amino acid 3-8, according to H3 numbering; SEQ IDNO: 37), the HA2 Helix A peptide DGVTNKVNS (amino acid 46-54; SEQ ID NO:38), the HA2 Helix B peptide MENERTLDFHDSNVK (amino acid 102-116; SEQ IDNO: 39) and the HA1 C-terminal peptide LVLATGLRNSP (amino acid 315-325;SEQ ID NO: 40). The binding region of FI28 variant 1 was different fromthat of FI6 since this antibody did not react with the HA1 C-terminalpeptide and the HA2 Helix B peptide.

Example 3 Generation and Characterization of FI6 Variants 3, 4 and 5with Improved Productivity

Several variants of FI6 variant 2 VH and VL were synthesized to improveproduction in mammalian cells and to remove unnecessary somaticmutations and unwanted features. The VH and VL genes were cloned intoexpression vectors encoding the constant region of IgG1 and CK,respectively. Germline sequences of FI6 variant 2 was determined withreference to the IMGT database. Antibody variants in which single ormultiple germline mutations were reverted to the germline were producedeither by synthesis (Genscript, Piscatawy, N.J.) or by site directedmutagenesis (Promega) and confirmed by sequencing. All variant sequenceswere codon optimized for expression in human cells using the GenScript's OptimumGene™ system. Monoclonal antibodies were produced by transienttransfection of suspension cultured 293 Freestyle cells (Invitrogen)with PEI. Supernatants from transfected cells were collected after 7days of culture and IgG were affinity purified by Protein Achromatography (GE Healthcare) and desalted against PBS. Productivity intransient expression system was evaluated in several independentexperiments. Mean values are shown in FIG. 1. As shown in FIG. 1, FI6variant 2 is produced at a titer of 13.5 μg/ml; FI6 variant 3 isproduced at a titer of 46.3 μg/ml; FI6 variant 4 is produced at a titerof 60.7 μg/ml; and FI6 variant 5 is produced at a titer of 61.6 μg/ml.Thus, we were able to achieve a 3.4-, 4.5- and 4.6-fold increase intiters of production of FI6 variants 3, 4 and 5, respectively, ascompared to FI6 variant 2.

The recombinant antibodies were also characterized for binding by ELISAto H5 and H7 HAs and neutralization of H5 and H7 pseudoviruses (Table 7)compared to the original FI6 variant 2 IgG. Half-area ELISA plates(Corning) were coated with 5 μl of 1 μg/ml baculovirus-derivedrecombinant HAs (Protein Sciences Corp.) in PBS. After blocking with 1%PBS/BSA, antibodies were added and the binding was revealed usingalkaline-phosphatase conjugated F(ab′)2 goat anti human IgG (SouthernBiotech). Plates were then washed, substrate (p-NPP, Sigma) was addedand plates were read at 405 nm. The relative affinities of antibodybinding to HAs were determined with ELISA by measuring the concentrationof antibody required to achieve 50% maximal binding (EC50). Forpseudovirus neutralization assays, serial dilutions of antibody wereincubated with a fixed concentration pseudovirus-containing culturesupernatants for 1 hour at 37° C. The mixtures were then added to HEK293T/17 cells and incubated for 3 days at 37° C. The cells were thenlysed with Britelite reagent (Perkin Elmer) and the relative light units(RLU) in the cell lysates were determined on a luminometer microplatereader (Veritas, Turner Biosystems). The reduction of infectivity wasdetermined by comparing the RLU in the presence and absence ofantibodies and expressed as percent neutralization. The 50% inhibitorydose (IC50) was defined as the sample concentration at which RLU werereduced 50% compared to virus control wells after subtraction ofbackground RLU in cell only control wells. Table 7 shows the FI6variants 3-5 that were selected based on improved sequencecharacteristics combined with preserved or improved binding activity.

TABLE 7 Neutralization Binding H5 A/VN/ H7 A/ck/FPV H5-HA H7-HA 1194/04Rostock/34 mAb ID EC₅₀ (μg/ml) EC₅₀ (μg/ml) IC50 (μg/ml) IC50 (μg/ml)FI6 variant 2 0.0145 0.0314 0.0054 0.0200 FI6 variant 3 0.0235 0.05730.0035 0.0274 FI6 variant 4 0.0137 0.0267 0.0035 0.0093 FI6 variant 50.0165 0.0473 0.0054 0.0220

FI6 variant 2 and FI6 variant 3 bound all recombinant or purified HAstested belonging to Group 1 (H1, H2, H5, H6, H8 and H9) and Group 2 (H3,H4, H7 and H10) with EC50 values ranging from 10 to 270 ng/ml (Table 8).In addition, FI6 variant 2 and FI6 variant 3 stained cells transfectedwith HA genes belonging to Group 1 (H11, H12, H13 and H16) and Group 2(H4, H10, H14 and H15 (Table 8).

TABLE 8 mAb ID FI6 FI6 HA protein variant 2 variant 3 ctr H1N1 A/SolomonIslands/3/   18⁽¹⁾  19⁽¹⁾   >20000⁽¹⁾ 06 H1N1 A/New Caledonia/20/ 1517 >20000 99 H1N1 A/California/04/09 17 17 >20000 H1N1 A/Brisbane/59/0715 17 >20000 H3N2 A/Wyoming/3/03 19 23 >20000 H3N2 A/New York/55/04 3238 >20000 H3N2 A/Brisbane/10/07 41 37 >20000 H5N1 A/Viet Nam/1203/04 1414 >20000 H5N1 A/Viet Nam/1/05 10 11 >20000 H7N7 A/Netherlands/219/03 2629 >20000 H9N2 A/Hong Kong/1073/03 14 15 >20000 H4N6 A/duck/ 273 185  >20000 Czechoslovakia/56 H6N5 A/shearwater/Australia/ 41 34 >200001/72 H7N3 A/Canada/444/04 33 25 >20000 H8N4 A/Alberta/357/84 3327 >20000 H10N4 A/mink/Sweded/3900/ 90 85 >20000 84 H13N6A/gull/Maryland/704/ 88 62 >20000 77 H2N2 A/Singapore/1/57 29 21 >20000H11N9 A/duck/Memphis/546/ +⁽²⁾ +⁽²⁾ — 74 H12N5 A/duck/Alberta/60/76 +⁽²⁾+⁽²⁾ — H13N6 A/gull/Maryland/704/ +⁽²⁾ +⁽²⁾ — 77 H16N3 A/black-headedgull/ +⁽²⁾ +⁽²⁾ — Sweded/2/99 H4N6 A/duck/ +⁽²⁾ +⁽²⁾ — Czechoslovakia/56H10N7 A/chicken/Germany/ +⁽²⁾ +⁽²⁾ — N49 H14N5 A/mallard/Astrakhan/ +⁽²⁾+⁽²⁾ — 263/82 H15N9 A/shearwater/West +⁽²⁾ +⁽²⁾ — Australia/2576/79B/Ohio/1/05 >20000   >20000   >20000 ⁽¹⁾EC50 values (ng/ml) as measuredby ELISA ⁽²⁾+ refers to positive staining of HA-transfected 293 cells

Example 4 Structural Characterization of FI6 Variant 3 Epitopes on H1and H3 HA

To identify the epitope recognized by FI6 variant 3 on Group 1 and Group2 HAs and to describe the molecular interactions between the antibodyand its target antigen, we crystallized complexes of FI6 variant 3 Fabfragment with H1 (Group 1) and H3 (Group 2) HA homotrimers. Forcrystallisation of FI6 variant 3/H1 homotrimeric HA complex, theectodomain of H1 HA0 was expressed in Sf9 insect cells. cDNAcorresponding to residues 11-329 (HA1) and 1-176 (HA2) (based on H3numbering) was cloned into a BioFocus expression vector incorporatingthe GP67 secretion signal to allow secretion of expressed protein intothe culture medium. The cloned cDNA was fused to a C-terminaltrimerizing foldon sequence to allow for the formation of the trimericform of H1 HA0. A thrombin cleavage sequence was included between thefoldon sequence and the C terminus of HA2 to allow for removal of thefoldon tag prior to crystallization and a 6-His tag was incorporated atthe extreme C-terminus of the expressed polypeptide sequence for use inaffinity purification.

Sf9 insect cells were infected with recombinant baculovirus and the6-His tagged H1 HA0 was recovered from the culture medium by passageover Ni-NTA resin (Qiagen) and gel filtration (S200 column). Elutedprotein corresponding to trimeric H1 HA0 was concentrated to 1 mg/mlbefore removal of the C-terminal tag by treatment with thrombin (5 unitsthrombin per mg HA0) for two hours at 20° C. Purified cleaved protein H1HA0 was finally fractionated on a Mono Q anion exchange column. To allowfor the formation of its complex with Fab-FI6 variant 3, purified H1 HA0at between 0.5 and 1 mg/ml was mixed with a two-fold molar excess ofpurified Fab-FI6 variant 3. The complex was allowed to form byincubation at 4° C. for three hours before separation from excessFab-FI6 variant 3 by fractionation on an S200 gel filtration column.

The purified complex of Fab-FI6 variant 3 and trimeric unprocessed H1HA0 was concentrated to 12 mg/ml for use in crystallization. Crystals ofthe complex of Fab-FI6 variant 3 and H1 HA0 were grown in hanging dropsby vapour diffusion over a well solution consisting of 0.1 M Bis Trispropane pH 7.0, 2.2 M ammonium sulfate. Crystals grew at 20° C. over aperiod of four weeks and were harvested from the drop into a 1:1 mix ofwell solution and 3.4 M sodium malonate pH 7.0 for cryo-protectionbefore flash freezing in liquid nitrogen. The data set was collected atthe Diamond Light Source, beam line IO3, and was indexed, integrated,and scaled using MOSFLM and SCALA, respectively.

Statistical analysis of the unit cell parameters and protein molecularweights suggested one haemagglutinin monomer and one Fab fragment perasymmetric unit; therefore, molecular replacement was performed usingsearch models in their monomeric states. Initial phases were obtainedusing coordinates of monomeric H1 HA (PDB ID 1RD8) as the search modelwith the CCP4 program PHASER. Using the automated model fitting programFFFEAR, the variable domain of the heavy chain was successfully fittedinto density and subsequent comparison with HA-antibody structures 3FKUand 3GBN allowed placement of the light chain variable domain.Alternating rounds of model building and refinement were performed usingCOOT and REFMAC5, respectively. This was repeated until as much of theelectron density map was fitted as possible and R-work and R-free valueshad leveled out.

Amino acids in the final PDB file are numbered following the Kabatconvention. The final model contains all of the H1 HA and the heavy andlight chain variable domains. For crystallization of FI6 variant 3/H3heterotrimeric HA complexes, X-31 (H3N2) virus and the bromelainreleased HA (BHA) were purified and the Fab fragments were prepared bypapain digestion. The FI6 variant 3 Fab was purified using Protein Asepharose affinity chromatography (HiTrap Protein A HP, 1 ml) followedby an S-200 size exclusion column. 3.5 mg of Fab were mixed with 3 mg ofX-31 BHA and incubated at 4° C. overnight for complex formation and thecomplex was purified using a superose 6 SEC column. Peak fractionscorresponding to the Fab-HA complex were pooled and concentrated forcrystallization.

FI6 variant 3-H3 complex crystals were grown by vapour diffusion insitting drops dispensed by an Oryx-6 crystallization robot from DouglasInstruments. Crystals were cryo-protected by the addition of 25%glycerol to the reservoir solution. The data set was collected at theDiamond Light Source, beam line IO3, and was indexed, integrated, andscaled using Denzo and Scalepack. The crystals, containing one H3 HAtrimer complexed with three FI6 variant 3 Fabs in the asymmetric unit,were solved by molecular replacement using Amore. The molecularreplacement calculations were done using the coordinates for the 2 Åstructure of H3 HA trimer, the heavy and light chain variable domains ofFI6 variant 3 from the FI6 variant 3/H1 complex and the constant domains(PDB ID 3HC0.pdb) as independent search objects. The molecularreplacement solution was refined with Refmac5 and Pheonix interspersedwith rounds of manual adjustments using Coot. Electron density maps weresubstantially improved by non-crystallographic averaging using DM.

X-ray crystallography showed that FI6 variant 3 bound to a conservedepitope in the F subdomain. Although the two HAs are phylogeneticallyand structurally distinct, and the complexes crystallize with differentpacking arrangements, the interaction surfaces were found to be verysimilar (FIG. 2, A and B). In both cases, each monomer of the HA trimerbinds one molecule of FI6 variant 3 (FIG. 3). The HCDR3 loop of FI6variant 3 binds into a shallow groove on the F subdomain of the HAswhere the sides of the groove are formed by residues from the A helix ofHA2, and parts of two strands of HA1 (38-42 and 318-320), whereas thebottom is formed by the HA2 turn encompassing residues 18-21 (FIG. 2, Aand B).

The HCDR3 loop crosses helix A, at an angle of about 45° , enablingLeu-100A, Tyr-100C, Phe-100D, and Trp-100F to make hydrophobic contactswith residues in the groove (FIG. 4). Tyr-100C and Trp-100F also makepotential hydrogen bonds with the side chain of Thr-318 of HA1 and themain chain carbonyl of residue 19 of HA1 respectively. Two additionalpolar interactions are formed by main chain carbonyls at residues 98 and99 of HCDR3 with Asn-53 and Thr-49 on helix A. Taken together theinteraction of HCDR3 with HA (H1 and H3) buries about 750 A2 of thesurface of the antibody and about ⅔ of this interaction is accounted forby the interaction with the HA2 chain.

Overall, the interactions made by FI6 variant 3 with the hydrophobicgroove on H1 and H3 are remarkably similar. The LCDR1 loop of FI6variant 3 makes two contacts with the side of helix A, opposite to theside that contributes to the hydrophobic groove; Phe-27D makeshydrophobic contact with the aliphatic part of Lys-39 and Asn-28hydrogen bonds to Asn-43, together accounting for a buried surface areaof about 190 Å2 for both H1 and H3. With H1 HA, which wasco-crystallized in the un-cleaved form, LCDR1 also makes extensivecontact with the un-cleaved “fusion peptide” of the neighboring distalright HA monomer (FIG. 3, B and C and Figure. 4, A and B), which amountsto an additional 320 Å2 of FI6 variant 3 buried surface.

Residues 28 and 29 of LCDR1 make main chain amide hydrogen bonds withthe main chain carbonyls of HA1 residue 329 and the next but oneresidue, Leu-2, of HA2, thus spanning the cleavage site. Phe-27D ofLCDR1 makes hydrophobic contacts with Leu-2 of the neighboring distalright HA2, while the side chain hydroxyl of Tyr-29 hydrogen bonds to themain chain carbonyl of residue 325 of the neighboring distal right HA1chain. In contrast to the very similar interaction between both H1 andH3 HAs with HCDR3, the interaction of LCDR1 with the “fusion peptide”from the neighboring cleaved distal right H3 HA monomer is significantlyless extensive than the interaction formed with the un-cleaved H1 HA(FIG. 3, insert B). Although Phe-27D again makes contact with thealiphatic moiety of Lys-39 of HA2, Tyr-29 makes potential hydrogen bondcontact with the main chain carbonyl of Ala-7 of the neighboring distalright HA2 (as opposed to residue 329 in un-cleaved H1 HA).

In contrast, there are no main chain contacts between the LCDR1 loop andthe “fusion peptide” of the cleaved H3 HA, accounting for the smallercontact area of 114 Å (cf 320 Å2 in H1 HA). It also seems that cleavageof the HA precursor to produce the H3 HA, results in the slightlydifferent orientation of FI6 variant 3 with respect to the HA in the FI6variant 3/H3 and FI6 variant 3 /H1 complexes. The contact residues atthe interface between FI6 variant 3 VH and VL chains and cleaved H3homotrimeric HA are reported in Table 9. The contact residues at theinterface between FI6 variant 3 VH and VL chains and uncleaved H1homotrimeric HA are reported in Table 10.

TABLE 9 Contact Residues at the Interface Between FI6 Variant 3 VH andVL and Cleaved H3-HA Trimer HA1 HA2 Cleavage site - Fusion peptideTrp-21 loop H3 T318 R321′ V323′ Q327′ S328′ R329′ G1′ L2′ F3′ G4′ A7′E11′ I18 D19 G20 W21 HK68 FI6 Y100c F100d F100d F100d F100d v3 W100f VHFI6 N28 Y29 Y29 Y29 v3 Y29 Y32 VK HA2 Helix A H3 L38 K39 T41 Q42 A43 I45D46 I48 N49 L52 N53 I56 E57 HK68 FI6 W100f W100f W100f L100b L98 Y52aL98 R99 v3 L100g Y100c R99 S100 VH S100h L100g S100 L100a FI6 R93 F27dF27d F27d v3 Y32 VK

TABLE 10 Contact Residues at the Interface Between FI6 Variant 3 VH andVL and Uncleaved H1-HA Trimer HA1 HA2 Cleavage site - Fusion peptideTrp-21 loop H1 T318 R321′ I323′ Q327′ S328′ R329′ G1′ L2′ F3′ G4′ A7′E11′ V18 D19 G20 W21 CA09 FI6 Y100c F100d F100d F100d Y100c v3 W100fF100d VH FI6 Y29 Y29 T27c T27c S27a F27d F27d v3 F27d T27c VK N28 F27dY29 N28 Y92 HA2 Helix A H1 L38 K39 T41 Q42 N43 I45 D46 I48 T49 V52 N53I56 E57 CA09 FI6 W100f W100f W100f L100a L100a R99 L98 R99 L98 v3 L100gY100c L100a R99 R99 VH S100h L100g FI6 R93 F27d F27d v3 N28 VK

The structures of two cross-reactive antibodies CR6261 and F10, whichare Group 1 specific, have previously been reported as complexes with H5and H1 HAs. The CR6261 and F10 antibodies binding to HA is mediated onlyby their VH domains which are oriented approximately the same as eachother with respect to the HA, but both antibodies are significantlyrotated relative to FI6 variant 3 and are 5-10 Å nearer to the membraneproximal end of HA (FIG. 3, D and E).

The structures of FI6 variant 3/H1 and FI6 variant 3/H3 presented herealso reveal that, although the binding sites on HA of the threeantibodies overlap extensively, the nature of the interactions made byFI6 variant 3 are markedly different to those made by CR6261 and F10antibodies. The most striking difference is that the interaction of FI6variant 3 with the hydrophobic groove on HA is mediated solely by thelong HCDR3, whereas for CR6261 and F10 all three HCDRs are involved inbinding.

An important difference between the FI6 variant 3/H1 and FI6 variant3/H3 complexes is that H3 HA is glycosylated at Asn-38 (HA1), as are H7,H10 and H15 HAs of Group 2, while H1, in common with all Group 1 HAs isnot. In the unbound structure of H3, this carbohydrate side-chainprojects from the beta strand of HA1 that contains the Asn-38 residue,towards helix A of HA2 of the same HA subunit, such that it wouldoverlap the footprint of FI6 variant 3 (FIG. 5A). Carbohydrateside-chains are known to influence the antigenicity of virusglycoproteins, therefore this overlap has been suggested to account forthe lack of binding to Group 2 HAs of other Group 1 cross-reactiveantibodies that target the membrane proximal region of HA. FI6 variant 3binding to H3 HA, however, is enabled by reorientation of theoligosaccharide, a rotation of about 80° away from the surface of theHA, so that it makes new contacts with Asp-53 and Asn-55 of the HCDR2loop (FIG. 5B).

Given that the flexibility of the carbohydrate side chain at Asn-38allows it to accommodate FI6 variant 3 binding to H3 HA, we askedwhether this glycosylation site was likely to be the reason that H3 HAdoes not bind to CR6261 or F10. Simple modeling suggests that the samechange in orientation of the carbohydrate side-chain would be compatiblewith the binding of the CR6261, but not the F10, cross-reactiveantibodies. The beta turn encompassing VH residues 73-77 of F10 wouldclash with the Asn-38-linked carbohydrate in the orientation it adoptsin the FI6 variant 3/H3 complex, and it is unclear whether thecarbohydrate would be free to rotate further out of the binding site toaccommodate F10 binding. However, neither CR6261 nor F10 were able toneutralize a H7 pseudovirus (A/chicken/Italy/99) in which theglycosylation site (Asn-38) was removed (IC50>50 μg/ml), indicating thatthe steric hindrance of the glycan is not the only structural constraintthat prevents binding of CR6261 and F10 antibodies to Group 2 HAs.

Besides the glycosylation of Asn-38 the most striking difference in theF subdomain structure between Group 1 and Group 2 HAs involves theGroup-distinctive environment and orientation of HA2 Trp-21. In Group-1HAs, Trp-21 is approximately parallel to the surface of the F subdomain,while in Group-2 HAs it is oriented roughly perpendicular to the surface(FIG. 5, C and D). All three antibodies (FI6 variant 3, CR6261 and F10)make contacts with Trp-21, mainly through a phenylalanine side chain;Phe-100D on FI6 variant 3, Phe-54 on CR6261 and Phe-55 on F10 (FIG. 5, Cand D). In the case of FI6 variant 3, local rearrangements in the HCDR3loop mean that Phe-100D sits approximately 2 Å deeper in the hydrophobicgroove in the H1 complex than it does in the H3 complex; it thusmaintains a similar contact distance with Trp-21 in both cases.

The two Group-1 specific antibodies position Phe-54 (CR6261) and Phe-55(F10) similarly to FI6 variant 3 in complex with H1 HA. However, asPhe-54 (CR6261) and Phe-55 (F10) are located on the short loop HCDR2,which connects two adjacent anti-parallel strands, it seems that thereis less flexibility than in FI6 variant 3 for the phenylalanine to movefurther out of the hydrophobic groove to accommodate the Group-2orientation of Trp-21. Thus, binding of CR6261 and F10 to Group 2 HAs islikely blocked by a steric clash between the HCDR2 phenylalanine andTrp-21.

In summary, the structural data obtained indicate that, although thecore epitope on helix A is similar to that recognized by CR6261 and F10,FI6 variant 3 binds with a different angle, 5-10 Å more membrane distaland contacts a larger area embracing helix A and extending to the fusionpeptide of the neighboring distal right monomer both in the cleaved anduncleaved forms (FIG. 6). FI6 variant 3 binding is mediated by both VHand VL CDRs, with prominent contributions of the long HCDR3, whichaccommodates different conformations of the Group-specific Trp-21 loop,and of the heavily mutated LCDR1. The use of both VH and VK chains andthe long HCDR3 are characteristic of naturally selected antibodies andcontrast with the property of phage-derived antibodies, such as CR6261and F10, which bind using only the VH chain. The contact residues in FI6variant 3 VH and VK are depicted in FIG. 7.

Example 5 In vivo Prophylactic Effect of FI6 Variant 3

The protective efficacy of FI6 variant 3 was tested in vivo in mousemodels of Influenza A virus infection. Groups of 6- to 8-week-old femaleBALB/c mice were injected intravenously (i.v.) with purified antibodiesat concentrations varying from 1 to 16 mg/kg. Three hours later, themice were deeply anaesthetized and challenged intranasally (i.n.) with10 MLD50 (fifty percent mouse lethal dose) of H1N1 A/PR/8/34. In atherapeutic setting, mice received the antibody 1, 2 or 3 days afterinfection. The mice were monitored daily for survival and weight lossuntil day 14 post-infection (p.i.). Animals that lost more than 25% oftheir initial body weight were euthanized in accordance with animalstudy protocol.

To evaluate the influence of FI6 variant 3 on viral replication, micechallenged with 10 MLD50 of H1N1 A/PR/8/34 received the antibody atdifferent time points and were sacrificed four days later to collectlungs and brains. The tissues were homogenized in Leibovitz L-15 medium(Invitrogen) supplemented with an antibiotic-antimycotic solution(Invitrogen) to achieve 10% w/v organ suspension. The organ homogenateswere titrated on MDCK cells and virus titers were determined. In aprophylactic setting FI6 variant 3 was fully protective and whenadministered at 4 mg/kg was partially protective (80% survival) whenadministered at 2 mg/kg to mice infected with Group 1 H1N1 A/PR/8/34virus (FIG. 8). Lung virus titers at day four after infection werereduced by approximately a hundred fold in mice treated with FI6 variant3 on day 0 or 1 day after infection (FIG. 9). In addition, FI6 variant 3prevented body weigh loss of mice infected with Group 2 H3N2 HK-x31virus (FIG. 8).

Example 6 Mechanisms of Virus Neutralization by FI6 Variant 3

For in vivo experiments aimed at determining the protective efficacy ofFI6 antibodies, we produced Fc mutants of FI6 variant 2 that lackcomplement binding (FI6-v2 KA) or complement and FcR binding (FI6-v2LALA). These antibodies showed the same binding and in vitroneutralizing properties as FI6 variant 2 and comparable half lives invivo (mean values 3.3, 3.4 and 3.5 days for FI6-v2, FI6-v2 KA and FI6-v2LALA, respectively). Their protective efficacy was tested in micelethally infected with A/Puerto Rico/8/34 (H1N1) virus. FI6.v2 fullyprotected mice from lethality when administered at 4 mg/kg and protected80% of mice at 2 mg/kg (FIG. 8F). When administered at 10 mg/kg, FI6-v2and FI6-v2 KA were fully protective, whereas FI6-v2 LALA showed asubstantial loss of activity, being able to protect only 40% of theanimals (FIG. 8F). This decreased efficacy was particularly evident whenmutant antibodies were administered at the limiting concentration of 3mg/kg (FIG. 8G).

To investigate mechanisms that contribute to the neutralizing activityof FI6 variant 3, NC/99 baculo-derived HA (Protein Sciences Corporation)were incubated for 40 minutes at 37° C. with a 15 times higher molaramounts FI6 variant 3, FE17, a non-specific mAb (HBD85) or no mAb in PBSsolution. TPCK treated trypsin was added to each sample to a finalconcentration of 2.5 μg/ml and digestion was performed at 37° C. for 5,10 and 20 minutes. At each time point, the digestion was stopped byadding a buffer containing SDS and DTT and by boiling it at 95° C. for 5minutes. Samples were then loaded on a 12% Tris-Glycine polyacrylamidegel. Protein transfer on a PVDF membrane was performed with the iBlotblotting system from Invitrogen. PVDF membrane was blocked for 30minutes with 10% non-fat dry milk in TBS-Tween. Incubation with primaryantibody against HA0 (in house produced biotinylated F032) was performedat 0.5 μg/ml in TBS-Tween overnight at 4° C. PVDF was washed three timeswith TBS-Tween and incubated for 1 h at RT with HRP-conjugatedStreptavidin (Sigma).

PVDF membrane was washed three times with TBS-Tween and positive bandsdetected using ECL Plus™ Western Blotting Detection Reagent (GEHealthcare) and the LAS4000 CCD camera system. The data in FIG. 10 showthat FI6 variant 3 inhibits cleavage of HA0 by TPCK-trypsin, indicatingthat the antibody light chain binding to unprocessed HA0blocksinfectivity, at least for those viruses where cleavage occursextracellularly.

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SEQ ID Number List SEQ ID NO Description Sequence  1 CDRH1 aa GFTFSTYA 2 CDRH2 aa ISYDGNYK  3 CDRH3 aa AKDSQLRSLLYFEWLSQGYFDP  4 CDRL1 aaQSVTFNYKNY  5 CDRL2 aa WAS  6 CDRL3 aa QQHYRTPPT  7 CDRH1 nucggattcacgttcagtacctatgcc  8 CDRH2 nuc atctcatacgatggaaattataaa  9CDRH3 nucgcgaaagactcccaactgcgatcactcctctattttgaatggttatcccagggatattttgacccc 10CDRL1 nuc cagagtgtcaccttcaactataagaactac 11 CDRL2 nuc tgggcatct 12CDRL3 nuc cagcaacattataggactcctccgacg 13 heavy ch aaQVQLVQSGGGVVQPGRSLRLSCVASGFTFSTYAMHWVRQAPGRGLEWVAVISYDGNYKYYADSVKGRFSISRDNSNSTLHLEMNTLRTEDTALYYCAKDSQLRSLLYFEWLSQGYFDPWGQGTLVTVTS 14 light ch aaDIQMTQSPDSLAVSLGARATINCKSSQSVTFNYKNYLAWYQQKPGQPPKVLIYWASARESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQHYRTPPTFGQGTKVEIK 15heavy ch nuccaggtgcagctggtgcagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgtagcctctggattcacgttcagtacctatgccatgcactgggtccgtcaggctccaggcagggggctggagtgggtggcagttatctcatacgatggaaattataaatactatgcagactctgtgaagggccgattctccatctccagagacaattccaacagcacgctgcatctagaaatgaacaccctgagaactgaggacacggctttatattactgtgcgaaagactcccaactgcgatcactcctctattttgaatggttatcccagggatattttgacccctggggccagggaacccttgtcaccgtcacctcag 16 light ch nucgacatccagatgacccagtctccagactccctggctgtatctctgggcgcgagggccaccatcaactgcaagtccagccagagtgtcaccttcaactataagaactacttagcttggtaccagcagaaaccaggacagcctcctaaagtgctcatttactgggcatctgcccgggaatcaggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggctgtttattactgtcagcaacattataggactcctccgacgttcggccaagggaccaaggtggagatcaaac 17 CDRH1 aa GFTFSNYG 18CDRH2 aa ISYDGSNK 19 CDRH3 aa AKERPLRLLRYFDWLSGGANDY 20 CDRL1 aaQSVLYSSNNKNY 21 CDRL2 aa WAS 22 CDRL3 aa QQYYRSPS 23 CDRH1 nucggattcaccttcagtaactatggc 24 CDRH2 nuc atatcatatgatggatctaataag 25CDRH3 nucgcgaaagagagaccccttcgcctattacgatattttgactggttatcggggggggcgaatgactac 26CDRL1 nuc cagagtgttttatacagctccaacaataagaactac 27 CDRL2 nuc tgggcatct 28CDRL3 nuc cagcagtattatagaagtccgtcc 29 heavy ch aaEVQLVESGGGAVQPGESLKLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKDTLYLQMNSLRAEDTALFYCAKERPLRLLRYFDWLSGGANDYWGQGTLVTVSS 30 light ch aaDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIDWASTRESGVPDRFSGSGSGTDFTLTISNLQVEDVAVYY CQQYYRSPSFGQGTKLEIK 31heavy ch nucgaggtgcagctggtggagtctgggggaggcgcggtccagcctggggagtccctgaaactctcctgtgcagcctctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaagggactggagtgggtggcagtcatatcatatgatggatctaataagtactatgcagactccgtgaagggccgattcaccatctccagagacaattccaaggacacgctgtatctgcaaatgaacagcctgagagctgaggacacggctctgttttactgtgcgaaagagagaccccttcgcctattacgatattttgactggttatcggggggggcgaatgactactggggccagggaaccctggtcaccgtctcctcag 32 light ch nucgacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcaagtccagccagagtgttttatacagctccaacaataagaactacttagcttggtaccagcagaaaccaggacagcctcctaagttgctcattgactgggcatctacccgggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcaatctgcaggttgaagatgtggccgtttattactgtcagcagtattatagaagtccgtcctttggccaggggaccaagctggagatcaaac 33 heavy ch aaQVQLVQSGGGVVQPGRSLRLSCVASGFTFSTYAMHWVRQAPGRGLEWVAVISYDGNYKYYADSVKGRFSISRDNSNNTLHLEMNTLRTEDTALYYCAKDSQLRSLLYFEWLSQGYFDPWGQGTLVTVTS 34 heavy ch nuccaggtgcagctggtgcagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgtagcctctggattcacgttcagtacctatgccatgcactgggtccgtcaggctccaggcagggggctggagtgggtggcagttatctcatacgatggaaattataaatactatgcagactctgtgaagggccgattctccatctccagagacaattccaacaacacgctgcatctagaaatgaacaccctgagaactgaggacacggctttatattactgtgcgaaagactcccaactgcgatcactcctctattttgaatggttatcccagggatattttgacccctggggccagggaaccctggtcaccgtcacctcag 35 heavy ch aaEVQLVESGGGAVQPGESLKLPCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKDTLYLQMNSLRAEDTALFYCAKERPLRLLRYFDWLSGGANDYWGQGTLVTVSS 36 heavy ch nucgaggtgcagctggtggagtctgggggaggcgcggtccagcctggggagtccctgaaactcccctgtgcagcctctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaagggactggagtgggtggcagtcatatcatatgatggatctaataagtactatgcagactccgtgaagggccgattcaccatctccagagacaattccaaggacacgctgtatctgcaaatgaacagcctgagagctgaggacacggctctgttttactgtgcgaaagagagaccccttcgcctattacgatattttgactggttatcggggggggcgaatgactactggggccagggaaccctggtcaccgtctcctcag 37 aa FGAIAG 38 aa DGVTNKVNS 39 aaMENERTLDFHDSNVK 40 aa LVLATGLRNSP 41 CDRH2 aa ISYDANYK 42 CDRH3 aaAKDSQLRSLLYFEWLSQGYFDY 43 CDRH3 aa AKDSQLRSLLYFEWLSQGYFEP 44 CDRL1 aaQSVTFNNKNY 45 CDRH1 nuc ggattcaccttttctacatacgct 46 CDRH2 nucatctcatacgacgctaactataag 47 CDRH3 nucgccaaagattctcagctgaggagtctgctgtatttcgaatggctgagccaggggtactttgattat 48CDRL1 nuc cagtctgtgactttcaactacaaaaattat 49 CDRL2 nuc tgggcttca 50CDRL3 nuc cagcagcactaccggactccacccacc 51 CDRH1 nucggattcactttttccacctacgca 52 CDRH2 nuc atctcatacgacgccaactataag 53CDRH3 nucgctaaggattctcagctgagaagtctgctgtattttgaatggctgtctcaggggtattttgaacct 54CDRL1 nuc cagtctgtgactttcaacaacaaaaattat 55 heavy ch aaQVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMHWVRQAPGKGLEWVAVISYDANYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSQLRSLLYFEWLSQGYFDYWGQGTLVTVSS 56 heavy ch nuccaggtgcagctggtggagtccggaggaggagtggtgcagccagggcggtctctgagactgagttgcgccgcttcaggattcaccttttctacatacgctatgcactgggtgcggcaggctcctggcaagggactggaatgggtggccgtgatctcatacgacgctaactataagtactatgccgatagcgtgaaaggcaggttcacaattagccgcgacaactccaagaatactctgtacctgcagatgaattccctgagggctgaggacaccgccgtgtactattgtgccaaagattctcagctgaggagtctgctgtatttcgaatggctgagccaggggtactttgattattggggacagggcactctggtgaccgtgagctcc 57 light ch aaDIVMTQSPDSLAVSLGERATINCKSSQSVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQHYRTPPTFGQGTKVEIK 58light ch nucgacatcgtgatgactcagtctcccgatagtctggccgtgtccctgggcgagagggctacaattaactgcaagagctcccagtctgtgactttcaactacaaaaattatctggcctggtaccagcagaagcctggacagccccctaaactgctgatctattgggcttcaacccgggaaagcggcgtgccagacagattctcaggcagcgggtccggaacagacttcaccctgacaatttctagtctgcaggccgaggacgtggccgtgtactattgtcagcagcactaccggactccacccacctttggccaggggacaaaggtggaaatcaaa 59 heavy ch aaQVQLVQSGGGVVQPGRSLRLSCVASGFTFSTYAMHWVRQAPGRGLEWVAVISYDANYKYYADSVKGRFSISRDNSQNTLHLEMNTLRTEDTALYYCAKDSQLRSLLYFEWLSQGYFEPWGQGTLVTVTS 60 heavy ch nuccaggtccagctggtccagagcggcggcggcgtggtccagccagggaggtcactgagactgtcatgcgtcgcttcaggattcactttttccacctacgcaatgcactgggtgcggcaggcacctggaagaggactggagtgggtggcagtcatctcatacgacgccaactataagtactatgctgatagcgtcaaaggcaggttcagcatttcccgcgacaacagtcagaatacactgcatctggagatgaataccctgcgaacagaagacactgccctgtactattgcgctaaggattctcagctgagaagtctgctgtattttgaatggctgtctcaggggtattttgaaccttgggggcagggcactctggtcaccgtcacttcc 61 light ch aaDIVMTQSPDSLAVSLGERATINCKSSQSVTFNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQHYRTPPTFGQGTKVEIK 62light ch nucgacatcgtgatgactcagtctcccgatagtctggccgtgtccctgggcgagagggctacaattaactgcaagagctcccagtctgtgactttcaacaacaaaaattatctggcctggtaccagcagaagcctggacagccccctaaactgctgatctattgggcttcaacccgggaaagcggcgtgccagacagattctcaggcagcgggtccggaacagacttcaccctgacaatttctagtctgcaggccgaggacgtggccgtgtactattgtcagcagcactaccggactccacccacctttggccaggggacaaaggtggaaatcaaa

1. An isolated antibody, or an antigen-binding fragment thereofcomprising a heavy chain variable region having at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NOs: 59 or55; and a light chain variable region having at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NOs: 57 or61, that neutralizes infection of a group 1 subtype and a group 2subtype of influenza A virus and specifically binds to an epitope in thestem region of an influenza A hemagglutinin (HA) trimer, and whereinsaid antibody, or antigen-binding fragment thereof, is produced intransfected cells at titers of at least 3 fold higher than the titer atwhich FI6 variant 2 is produced.
 2. An isolated antibody, or anantigen-binding fragment thereof of claim 1, comprising a heavy chainvariable region having at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 55; and a light chain variableregion having at least 90% sequence identity to the amino acid sequenceas set forth in SEQ ID NO: 57, that neutralizes infection of a group 1subtype and a group 2 subtype of influenza A virus and specificallybinds to an epitope in the stem region of an influenza A hemagglutinin(HA) trimer, and wherein said antibody, or antigen-binding fragmentthereof, is produced in transfected cells at titers of at least 3 foldhigher than the titer at which FI6 variant 2 is produced.
 3. An isolatedantibody, or an antigen-binding fragment thereof of claim 1, comprisinga heavy chain variable region having at least 90% sequence identity tothe amino acid sequence as set forth in SEQ ID NO: 59; and a light chainvariable region having at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 57, that neutralizes infection of agroup 1 subtype and a group 2 subtype of influenza A virus andspecifically binds to an epitope in the stem region of an influenza Ahemagglutinin (HA) trimer, and wherein said antibody, or antigen-bindingfragment thereof, is produced in transfected cells at titers of at least3 fold higher than the titer at which FI6 variant 2 is produced.
 4. Anisolated antibody, or an antigen-binding fragment thereof of claim 1,comprising a heavy chain variable region having at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 59; and alight chain variable region having at least 90% sequence identity to theamino acid sequence as set forth in SEQ ID NO: 61, that neutralizesinfection of a group 1 subtype and a group 2 subtype of influenza Avirus and specifically binds to an epitope in the stem region of aninfluenza A hemagglutinin (HA) trimer, and wherein said antibody, orantigen-binding fragment thereof, is produced in transfected cells attiters of at least 3 fold higher than the titer at which FI6 variant 2is produced.
 5. The isolated antibody, or an antigen-binding fragmentthereof of claim 1, that neutralizes infection of a group 1 subtype anda group 2 subtype of influenza A virus and comprises: (i) the heavychain CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NOs: 1, 41and 43, respectively, or as set forth in SEQ ID NOs: 1, 41 and 42,respectively; and (ii) the light chain CDR1, CDR2, and CDR3 sequences asset forth in SEQ ID NOs: 4, 5 and 6, respectively, or as set forth inSEQ ID NOs: 44, 5 and 6, respectively.
 6. The isolated antibody, orantigen binding fragment thereof of claim 1, wherein the antibody is ahuman antibody, a monoclonal antibody, a purified antibody, a singlechain antibody, Fab, Fab′, F(ab′)2, Fv or scFv.
 7. The isolatedantibody, or antigen-binding fragment thereof of claim 1, wherein saidantibody, or antigen-binding fragment thereof, specifically binds to aninfluenza A HA of subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11,H12, H13, H14, H15 and H16.
 8. A nucleic acid molecule comprising apolynucleotide encoding the antibody, or an antigen binding fragmentthereof, of claim
 1. 9. A vector comprising the nucleic acid molecule ofclaim
 8. 10. A cell expressing the antibody or an antigen bindingfragment thereof of claim
 1. 11. A pharmaceutical composition comprisingthe antibody or antigen-binding fragment of claim 1, and apharmaceutically acceptable diluent or carrier.
 12. A method fordiagnosing influenza A virus infection in a subject, the methodcomprising contacting the antibody or antigen-binding fragment thereofof claim 1 with a sample from the subject.
 13. A method for monitoringthe quality of anti-influenza A virus vaccines, comprising contactingthe vaccine with the antibody or antigen-binding fragment thereof ofclaim 1 and determining whether the vaccine contains a specific epitopein correct conformation.
 14. A method of reducing influenza A virusinfection, or lowering the risk of influenza A virus infection,comprising: administering to a subject in need thereof, aprophylactically or therapeutically effective amount of the antibody orantigen binding fragment thereof of claim
 1. 15. An isolated antibody,or an antigen-binding fragment thereof comprising a heavy chain variableregion having at least 90% sequence identity to the amino acid sequenceas set forth in SEQ ID NOs: 59 or 55; and a light chain variable regionhaving at least 90% sequence identity to the amino acid sequence as setforth in SEQ ID NOs: 57 or 61, that neutralizes infection of a group 1subtype and a group 2 subtype of influenza A virus and specificallybinds to an epitope in the stem region of an influenza A hemagglutinin(HA) trimer, wherein the heavy and light chain of said antibody, orantigen binding fragment thereof, contact amino acids in a first,proximal monomer and a second, distal, right monomer of said HA trimer,and wherein said antibody, or antigen-binding fragment thereof, isproduced in transfected cells at titers of at least 3 fold higher thanthe titer at which FI6 variant 2 is produced, in which: a. the heavychain of said antibody, or antigen-binding fragment thereof, contactsthe amino acid at position 318 in HA1 and amino acid residues atpositions 18, 19, 20, 21, 38, 41, 42, 45, 49, 53, and 57 in HA2 of saidfirst or second monomer, and wherein said monomer is uncleaved orcleaved; b. the light chain of said antibody, or antigen-bindingfragment thereof, contacts amino acid residues at positions 38, 39, and43 in HA2 of said proximal monomer, and amino acid residues at positions327, 328, and 329 in HA1 and 1, 2, 3, and 4 in HA2 of said distal rightmonomer, and wherein said proximal and said distal right monomers areuncleaved; c. the light chain of said antibody, or antigen-bindingfragment thereof, contacts amino acid residues at positions 38, 39, 42,and 46 in HA2 of said proximal monomer and amino acid residues atpositions 321 and 323 in HA1 and 7 and 11 in HA2 of said distal rightmonomer, and wherein said proximal and said distal right monomers arecleaved; d. the antibody, or antigen-binding fragment thereofspecifically binds to an epitope that comprises the amino acid atposition 318 of HA1 and amino acid residues at positions 18, 19, 20, 21,38, 39, 41, 42, 43, 45, 48, 49, 53, 56, and 57 of HA2 of said proximalmonomer, and the amino acid residues at positions 327, 328, 329 of HA1and amino acid residues at positions 1, 2, 3, and 4 of HA2 polypeptideof said distal right monomer, wherein said proximal and said distalright monomers are uncleaved; e. the antibody, or antigen-bindingfragment thereof specifically binds to an epitope that comprises theamino acid at position 318 of HA1 and the amino acid residues atpositions 18, 19, 20, 21, 38, 39, 41, 42, 45, 46, 49, 52, 53, and 57 ofHA2 of said proximal monomer, and the amino acid residues at positions321 and 323 of HA1 and amino acid residues at positions 7 and 11 of HA2of said distal right monomer, wherein said proximal and said distalright monomers are cleaved; or the antibody, or f. the antigen-bindingfragment thereof specifically binds to an epitope that comprises theamino acid at position 329 of HA1 and the amino acid residues atpositions 1, 2, 3, and 4 of HA2, wherein said HA1 and HA2 are present inan uncleaved monomer of said HA trimer.
 16. The isolated antibody, or anantigen-binding fragment thereof of claim 15, that neutralizes infectionof a group 1 subtype and a group 2 subtype of influenza A virus andcomprises: (i) the heavy chain CDR1, CDR2 and CDR3 sequences as setforth in SEQ ID NOs: 1, 41 and 43, respectively, or as set forth in SEQID NOs: 1, 41 and 42, respectively; and (ii) the light chain CDR1, CDR2,and CDR3 sequences as set forth in SEQ ID NOs: 4, 5 and 6, respectively,or as set forth in SEQ ID NOs: 44, 5 and 6, respectively.
 17. Theantibody, or antigen binding fragment thereof of claim 15, wherein theantibody is a human antibody, a monoclonal antibody, a purifiedantibody, a single chain antibody, Fab, Fab′, F(ab′)2, Fv or scFv. 18.The antibody, or antigen-binding fragment thereof of claim 15, whereinsaid antibody, or antigen-binding fragment thereof, specifically bindsto an influenza A HA of subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9,H10, H11, H12, H13, H14, H15 and H16.